Isolated human kinase proteins

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.09/801,861, filed on Mar. 9, 2001 and issued on Dec. 10, 2002 as U.S.Pat. No. 6,492,154, which claims benefit of U.S. Provisional ApplicationNo. 60/265,151, filed on Jan. 31, 2001.

FIELD OF THE INVENTION

The present invention is in the field of kinase proteins that arerelated to the Pftaire kinase subfamily, recombinant DNA molecules, andprotein production. The present invention specifically provides novelpeptides and proteins, representing two alternative splice forms, thateffect protein phosphorylation and nucleic acid molecules encoding suchpeptide and protein molecules, all of which are useful in thedevelopment of human therapeutics and diagnostic compositions andmethods.

BACKGROUND OF THE INVENTION

Protein Kinases

Kinases regulate many different cell proliferation, differentiation, andsignaling processes by adding phosphate groups to proteins. Uncontrolledsignaling has been implicated in a variety of disease conditionsincluding inflammation, cancer, arteriosclerosis, and psoriasis.Reversible protein phosphorylation is the main strategy for controllingactivities of eukaryotic cells. It is estimated that more than 1000 ofthe 10,000 proteins active in a typical mammalian cell arephosphorylated. The high energy phosphate, which drives activation, isgenerally transferred from adenosine triphosphate molecules (ATP) to aparticular protein by protein kinases and removed from that protein byprotein phosphatases. Phosphorylation occurs in response toextracellular signals (hormones, neurotransmitters, growth anddifferentiation factors, etc), cell cycle checkpoints, and environmentalor nutritional stresses and is roughly analogous to turning on amolecular switch. When the switch goes on, the appropriate proteinkinase activates a metabolic enzyme, regulatory protein, receptor,cytoskeletal protein, ion channel or pump, or transcription factor.

The kinases comprise the largest known protein group, a superfamily ofenzymes with widely varied functions and specificities. They are usuallynamed after their substrate, their regulatory molecules, or some aspectof a mutant phenotype. With regard to substrates, the protein kinasesmay be roughly divided into two groups; those that phosphorylatetyrosine residues (protein tyrosine kinases, PTK) and those thatphosphorylate serine or threonine residues (serine/threonine kinases,STK). A few protein kinases have dual specificity and phosphorylatethreonine and tyrosine residues. Almost all kinases contain a similar250-300 amino acid catalytic domain. The N-terminal domain, whichcontains subdomains I-IV, generally folds into a two-lobed structure,which binds and orients the ATP (or GTP) donor molecule. The larger Cterminal lobe, which contains subdomains VI A-XI, binds the proteinsubstrate and carries out the transfer of the gamma phosphate from ATPto the hydroxyl group of a serine, threonine, or tyrosine residue.Subdomain V spans the two lobes.

The kinases may be categorized into families by the different amino acidsequences (generally between 5 and 100 residues) located on either sideof, or inserted into loops of, the kinase domain. These added amino acidsequences allow the regulation of each kinase as it recognizes andinteracts with its target protein. The primary structure of the kinasedomains is conserved and can be further subdivided into 11 subdomains.Each of the 11 subdomains contains specific residues and motifs orpatterns of amino acids that are characteristic of that subdomain andare highly conserved (Hardie, G. and Hanks, S. (1995) The Protein KinaseFacts Books, Vol I:7-20 Academic Press, San Diego, Calif.).

The second messenger dependent protein kinases primarily mediate theeffects of second messengers such as cyclic AMP (cAMP), cyclic GMP,inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate,cyclic-ADPribose, arachidonic acid, diacylglycerol andcalcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) areimportant members of the STK family. Cyclic-AMP is an intracellularmediator of hormone action in all prokaryotic and animal cells that havebeen studied. Such hormone-induced cellular responses include thyroidhormone secretion, cortisol secretion, progesterone secretion, glycogenbreakdown, bone resorption, and regulation of heart rate and force ofheart muscle contraction. PKA is found in all animal cells and isthought to account for the effects of cyclic-AMP in most of these cells.Altered PKA expression is implicated in a variety of disorders anddiseases including cancer, thyroid disorders, diabetes, atherosclerosis,and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison'sPrinciples of Internal Medicine, McGraw-Hill, New York, N.Y., pp.416-431, 1887).

Calcium-calmodulin (CaM) dependent protein kinases are also members ofSTK family. Calmodulin is a calcium receptor that mediates many calciumregulated processes by binding to target proteins in response to thebinding of calcium. The principle target protein in these processes isCaM dependent protein kinases. CaM-kinases are involved in regulation ofsmooth muscle contraction (MLC kinase), glycogen breakdown(phosphorylase kinase), and neurotransmission (CaM kinase I and CaMkinase II). CaM kinase I phosphorylates a variety of substratesincluding the neurotransmitter related proteins synapsin I and II, thegene transcription regulator, CREB, and the cystic fibrosis conductanceregulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal14:3679-86). CaM II kinase also phosphorylates synapsin at differentsites, and controls the synthesis of catecholamines in the brain throughphosphorylation and activation of tyrosine hydroxylase. Many of the CaMkinases are activated by phosphorylation in addition to binding to CaM.The kinase may autophosphorylate itself, or be phosphorylated by anotherkinase as part of a “kinase cascade”.

Another ligand-activated protein kinase is 5′-AMP-activated proteinkinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81).Mammalian AMPK is a regulator of fatty acid and sterol synthesis throughphosphorylation of the enzymes acetyl-CoA carboxylase andhydroxymethylglutaryl-CoA reductase and mediates responses of thesepathways to cellular stresses such as heat shock and depletion ofglucose and ATP. AMPK is a heterotrimeric complex comprised of acatalytic alpha subunit and two non-catalytic beta and gamma subunitsthat are believed to regulate the activity of the alpha subunit.Subunits of AMPK have a much wider distribution in non-lipogenic tissuessuch as brain, heart, spleen, and lung than expected. This distributionsuggests that its role may extend beyond regulation of lipid metabolismalone.

The mitogen-activated protein kinases (MAP) are also members of the STKfamily. MAP kinases also regulate intracellular signaling pathways. Theymediate signal transduction from the cell surface to the nucleus viaphosphorylation cascades. Several subgroups have been identified, andeach manifests different substrate specificities and responds todistinct extracellular stimuli (Egan, S. E. and Weinberg, R. A. (1993)Nature 365:781-783). MAP kinase signaling pathways are present inmammalian cells as well as in yeast. The extracellular stimuli thatactivate mammalian pathways include epidermal growth factor (EGF),ultraviolet light, hyperosmolar medium, heat shock, endotoxiclipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumornecrosis factor (TNF) and interleukin-1 (IL-1).

PRK (proliferation-related kinase) is a serum/cytokine inducible STKthat is involved in regulation of the cell cycle and cell proliferationin human megakaroytic cells (Li, B. et al. (1996) J. Biol. Chem. 271:19402-8). PRK is related to the polo (derived from humans polo gene)family of STKs implicated in cell division. PRK is downregulated in lungtumor tissue and may be a proto-oncogene whose deregulated expression innormal tissue leads to oncogenic transformation. Altered MAP kinaseexpression is implicated in a variety of disease conditions includingcancer, inflammation, immune disorders, and disorders affecting growthand development.

The cyclin-dependent protein kinases (CDKs) are another group of STKsthat control the progression of cells through the cell cycle. Cyclinsare small regulatory proteins that act by binding to and activating CDKsthat then trigger various phases of the cell cycle by phosphorylatingand activating selected proteins involved in the mitotic process. CDKsare unique in that they require multiple inputs to become activated. Inaddition to the binding of cyclin, CDK activation requires thephosphorylation of a specific threonine residue and thedephosphorylation of a specific tyrosine residue.

Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosineresidues on their target proteins and may be divided into transmembrane,receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembraneprotein-tyrosine kinases are receptors for most growth factors. Bindingof growth factor to the receptor activates the transfer of a phosphategroup from ATP to selected tyrosine side chains of the receptor andother specific proteins. Growth factors (GF) associated with receptorPTKs include; epidermal GF, platelet-derived GF, fibroblast GF,hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascularendothelial GF, and macrophage colony stimulating factor.

Non-receptor PTKs lack transmembrane regions and, instead, formcomplexes with the intracellular regions of cell surface receptors. Suchreceptors that function through non-receptor PTKs include those forcytokines, hormones (growth hormone and prolactin) and antigen-specificreceptors on T and B lymphocytes.

Many of these PTKs were first identified as the products of mutantoncogenes in cancer cells where their activation was no longer subjectto normal cellular controls. In fact, about one third of the knownoncogenes encode PTKs, and it is well known that cellular transformation(oncogenesis) is often accompanied by increased tyrosine phosphorylationactivity (Carbonneau H and Tonks NK (1992) Annu. Rev. Cell. Biol.8:463-93). Regulation of PTK activity may therefore be an importantstrategy in controlling some types of cancer.

Pftaire Protein Kinases

The novel human proteins, and encoding gene, provided by the presentinvention are related to Pftaire serine/threonine kinases. Specifically,two alternative splice forms of the same gene are provided by thepresent invention, referred to herein as “splice form 1” and “spliceform 2”. The sequences of a cDNA molecule encoding splice form 1 and atranscript sequence encoding splice form 2 are provided in FIG. 1. Theamino acid sequences of each splice form are provided in FIG. 2; spliceform 1 is 343 amino acids in length and splice form 2 is 435 amino acidsin length.

The proteins of the present invention are similar to Pftaire-1previously isolated from the mouse (Besset et al., Mol Reprod Dev 1998May;50(1):18-29 and Lazzaro et al., J Neurochem 1997 July;69(1):348-64)and human (Nagase et al., DNA Res Dec. 31, 1998; 5(6):355-64). Pftairekinases are related to Cdk and cdc2 kinases, which are expressed in thebrain and other mitotic tissues; however, Pftaire expression patterns inthe nervous system differ from those of Cdk and cdc2 kinases and Pftairekinases are likely to have distinct functions (Lazzaro et al., JNeurochem 1997 July;69(1):348-64).

Mouse Pftaire-1 shares 50% and 49% amino acid identity with Cdk5 andPctaire-3, respectively. Two transcripts, approximately 5.5 and 4.9 kbin size, have been detected. These transcripts are highly expressed inthe brain, testis and embryo, and expressed at low levels in all otheranalyzed tissues in the mouse. Pftaire-1 is expressed in late pachytenespermatocytes in the testis and in post-mitotic neuronal cells in boththe brain and embryo, suggesting that Pftaire-1 plays key roles inmeiosis and neuron differentiation and/or function (Besset et al., MolReprod Dev 1998 May;50(1):18-29).

Pftaire is highly expressed in both postnatal and adult nervous tissue.Certain terminally differentiated neurons and neuroglia have been shownto express Pftaire mRNA and proteins. Pftaire proteins are found in thenucleus and cytoplasm of neuron cells. These expression patterns suggestthat Pftaire kinases play key roles in regulating and maintaining thepostmitotic and differentiated condition of nervous system cells(Lazzaro et al., J Neurochem 1997 July;69(1):348-64).

Kinase proteins, particularly members of the Pftaire kinase subfamily,are a major target for drug action and development. Accordingly, it isvaluable to the field of pharmaceutical development to identify andcharacterize previously unknown members of this subfamily of kinaseproteins. The present invention advances the state of the art byproviding previously unidentified human kinase proteins that havehomology to members of the Pftaire kinase subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human kinase peptides and proteins, representing twoalternative splice forms, that are related to the Pftaire kinasesubfamily, as well as allelic variants and other mammalian orthologsthereof. These unique peptide sequences, and nucleic acid sequences thatencode these peptides, can be used as models for the development ofhuman therapeutic targets, aid in the identification of therapeuticproteins, and serve as targets for the development of human therapeuticagents that modulate kinase activity in cells and tissues that expressthe kinase. Experimental data as provided in FIG. 1 indicates expressionin humans in uterus endometrium adenocarcinoma, testis, lungfibroblasts, kidney renal cell adenocarcinoma, and the brain.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequences of a cDNA molecule (for spliceform 1; SEQ ID NO:1) and a transcript sequence (for splice form 2; SEQID NO:4) that encode the kinase proteins of the present invention. Inaddition, structure and functional information is provided, such as ATGstart, stop and tissue distribution, where available, that allows one toreadily determine specific uses of the inventions based on thesemolecular sequences. Experimental data as provided in FIG. 1 indicatesexpression in humans in uterus endometrium adenocarcinoma, testis, lungfibroblasts, kidney renal cell adenocarcinoma, and the brain.

FIG. 2 provides the predicted amino acid sequence of splice form 1 (SEQID NO:2) and splice form 2 (SEQ ID NO:5) of the kinase of the presentinvention. In addition structure and functional information such asprotein family, function, and modification sites is provided whereavailable, allowing one to readily determine specific uses of theinventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding the kinaseproteins of the present invention. (SEQ ID NO:3) In addition structureand functional information, such as intron/exon structure, promoterlocation, etc., is provided where available, allowing one to readilydetermine specific uses of the inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 26 differentnucleotide positions (SNPs were also identified at an additional 30nucleotide positions 3′ of the ORF, as provided in U.S. Ser. No.60/265,151, filed Jan. 31, 2001).

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a kinase protein or part of a kinase protein and arerelated to the Pftaire kinase subfamily. Utilizing these sequences,additional genomic sequences were assembled and transcript and/or cDNAsequences were isolated and characterized. Based on this analysis, thepresent invention provides amino acid sequences of human kinase peptidesand proteins, representing two alternative splice forms (referred toherein as “splice form 1” and “splice form 2”), that are related to thePftaire kinase subfamily, nucleic acid sequences in the form oftranscript sequences, cDNA sequences and/or genomic sequences thatencode these kinase peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the kinase of the present invention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known kinase proteins of the Pftaire kinasesubfamily and the expression pattern observed. Experimental data asprovided in FIG. 1 indicates expression in humans in uterus endometriumadenocarcinoma, testis, lung fibroblasts, kidney renal celladenocarcinoma, and the brain. The art has clearly established thecommercial importance of members of this family of proteins and proteinsthat have expression patterns similar to that of the present gene. Someof the more specific features of the peptides of the present invention,and the uses thereof, are described herein, particularly in theBackground of the Invention and in the annotation provided in theFigures, and/or are known within the art for each of the known Pftairefamily or subfamily of kinase proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thekinase family of proteins and are related to the Pftaire kinasesubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the kinase peptides of the present invention, kinase peptides,or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the kinase peptides disclosed in the FIG. 2, (encoded bythe nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thekinase peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

The isolated kinase peptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods.Experimental data as provided in FIG. 1 indicates expression in humansin uterus endometrium adenocarcinoma, testis, lung fibroblasts, kidneyrenal cell adenocarcinoma, and the brain. For example, a nucleic acidmolecule encoding the kinase peptide is cloned into an expressionvector, the expression vector introduced into a host cell and theprotein expressed in the host cell. The protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Many of these techniques are describedin detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NOS:2 and 5), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NOS:1 and 4) and the genomic sequences providedin FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein isprovided in FIG. 2. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NOS:2 and 5), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NOS:1 and 4) and the genomic sequences providedin FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NOS:2 and 5), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NOS:1 and 4) and the genomic sequences provided in FIG. 3(SEQ ID NO:3). A protein comprises an amino acid sequence when the aminoacid sequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the kinase peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

The kinase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a kinase peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the kinase peptide. “Operatively linked”indicates that the kinase peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the kinase peptide.

In some uses, the fusion protein does not affect the activity of thekinase peptide per se. For example, the fusion protein can include, butis not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant kinase peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A kinase peptide-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the kinase peptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the kinase peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thekinase peptides of the present invention as well as being encoded by thesame genetic locus as the kinase peptide provided herein. The geneencoding the novel kinase protein of the present invention is located ona genome component that has been mapped to human chromosome 2 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data.

Allelic variants of a kinase peptide can readily be identified as beinga human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the kinase peptide as well asbeing encoded by the same genetic locus as the kinase peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. The gene encoding the novel kinase protein of thepresent invention is located on a genome component that has been mappedto human chromosome 2 (as indicated in FIG. 3), which is supported bymultiple lines of evidence, such as STS and BAC map data. As usedherein, two proteins (or a region of the proteins) have significanthomology when the amino acid sequences are typically at least about70-80%, 80-90%, and more typically at least about 90-95% or morehomologous. A significantly homologous amino acid sequence, according tothe present invention, will be encoded by a nucleic acid sequence thatwill hybridize to a kinase peptide encoding nucleic acid molecule understringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in the geneencoding the kinase protein of the present invention. SNPs wereidentified at 26 different nucleotide positions (SNPs were alsoidentified at an additional 30 nucleotide positions 3′ of the ORF, asprovided in U.S. Ser. No. 60/265,151 filed Jan. 31, 2001). Some of theseSNPs, which are located outside the ORF and in introns, may affectcontrol/regulatory elements.

Paralogs of a kinase peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the kinase peptide, as being encoded by a gene from humans, and ashaving similar activity or function. Two proteins will typically beconsidered paralogs when the amino acid sequences are typically at leastabout 60% or greater, and more typically at least about 70% or greaterhomology through a given region or domain. Such paralogs will be encodedby a nucleic acid sequence that will hybridize to a kinase peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a kinase peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the kinase peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from mammals, preferablyprimates, for the development of human therapeutic targets and agents.Such orthologs will be encoded by a nucleic acid sequence that willhybridize to a kinase peptide encoding nucleic acid molecule undermoderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the kinase peptides of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the kinase peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a kinase peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

Variant kinase peptides can be fully functional or can lack function inone or more activities, e.g. ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variation orvariation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as kinase activity or in assays such as an in vitro proliferativeactivity. Sites that are critical for binding partner/substrate bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance or photoaffinity labeling (Smith et al., J.Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312(1992)).

The present invention further provides fragments of the kinase peptides,in addition to proteins and peptides that comprise and consist of suchfragments, particularly those comprising the residues identified in FIG.2. The fragments to which the invention pertains, however, are not to beconstrued as encompassing fragments that may be disclosed publicly priorto the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a kinase peptide. Such fragments canbe chosen based on the ability to retain one or more of the biologicalactivities of the kinase peptide or could be chosen for the ability toperform a function, e.g. bind a substrate.or act as an immunogen.Particularly important fragments are biologically active fragments,peptides that are, for example, about 8 or more amino acids in length.Such fragments will typically comprise a domain or motif of the kinasepeptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in kinase peptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art (someof these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the kinase peptides of the present invention also encompassderivatives or analogs in which a substituted amino acid residue is notone encoded by the genetic code, in which a substituent group isincluded, in which the mature kinase peptide is fused with anothercompound, such as a compound to increase the half-life of the kinasepeptide (for example, polyethylene glycol), or in which the additionalamino acids are fused to the mature kinase peptide, such as a leader orsecretory sequence or a sequence for purification of the mature kinasepeptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theFigures; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a kinase-effectorprotein interaction or kinase-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, kinases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the kinase. Experimental data as providedin FIG. 1 indicates that the kinase proteins of the present inventionare expressed in humans in uterus endometrium adenocarcinoma, testis,lung fibroblasts, and kidney renal cell adenocarcinoma, as indicated byvirtual northern blot analysis. In addition, tissue-screening panelsindicate expression in the brain. A large percentage of pharmaceuticalagents are being developed that modulate the activity of kinaseproteins, particularly members of the Pftaire subfamily (see Backgroundof the Invention). The structural and functional information provided inthe Background and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in humans in uterus endometriumadenocarcinoma, testis, lung fibroblasts, kidney renal celladenocarcinoma, and the brain. Such uses can readily be determined usingthe information provided herein, that which is known in the art, androutine experimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to kinases that are related to members ofthe Pftaire subfamily. Such assays involve any of the known kinasefunctions or activities or properties useful for diagnosis and treatmentof kinase-related conditions that are specific for the subfamily ofkinases that the one of the present invention belongs to, particularlyin cells and tissues that express the kinase. Experimental data asprovided in FIG. 1 indicates that the kinase proteins of the presentinvention are expressed in humans in uterus endometrium adenocarcinoma,testis, lung fibroblasts, and kidney renal cell adenocarcinoma, asindicated by virtual northern blot analysis. In addition,tissue-screening panels indicate expression in the brain.

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the kinase, as a biopsy orexpanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in uterus endometrium adenocarcinoma,testis, lung fibroblasts, kidney renal cell adenocarcinoma, and thebrain. In an alternate embodiment, cell-based assays involve recombinanthost cells expressing the kinase protein.

The polypeptides can be used to identify compounds that modulate kinaseactivity of the protein in its natural state or an altered form thatcauses a specific disease or pathology associated with the kinase. Boththe kinases of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the kinase. These compounds can befurther screened against a functional kinase to determine the effect ofthe compound on the kinase activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the kinase to a desired degree.

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between thekinase protein and a molecule that normally interacts with the kinaseprotein, e.g. a substrate or a component of the signal pathway that thekinase protein normally interacts (for example, another kinase). Suchassays typically include the steps of combining the kinase protein witha candidate compound under conditions that allow the kinase protein, orfragment, to interact with the target molecule, and to detect theformation of a complex between the protein and the target or to detectthe biochemical consequence of the interaction with the kinase proteinand the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantkinases or appropriate fragments containing mutations that affect kinasefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) kinase activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate kinase activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the kinase proteindependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the kinasecan be used as an endpoint assay. These include all of the biochemicalor biochemical/biological events described herein, in the referencescited herein, incorporated by reference for these endpoint assaytargets, and other functions known to those of ordinary skill in the artor that can be readily identified using the information provided in theFigures, particularly FIG. 2. Specifically, a biological function of acell or tissues that expresses the kinase can be assayed. Experimentaldata as provided in FIG. 1 indicates that the kinase proteins of thepresent invention are expressed in humans in uterus endometriumadenocarcinoma, testis, lung fibroblasts, and kidney renal celladenocarcinoma, as indicated by virtual northern blot analysis. Inaddition, tissue-screening panels indicate expression in the brain.

Binding and/or activating compounds can also be screened by usingchimeric kinase proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native kinase. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the kinase is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the kinase (e.g. binding partners and/or ligands). Thus, a compoundis exposed to a kinase polypeptide under conditions that allow thecompound to bind or to otherwise interact with the polypeptide. Solublekinase polypeptide is also added to the mixture. If the test compoundinteracts with the soluble kinase polypeptide, it decreases the amountof complex formed or activity from the kinase target. This type of assayis particularly useful in cases in which compounds are sought thatinteract with specific regions of the kinase. Thus, the solublepolypeptide that competes with the target kinase region is designed tocontain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the kinase protein, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofkinase-binding protein found in the bead fraction quantitated from thegel using standard electrophoretic techniques. For example, either thepolypeptide or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies reactive with the protein but whichdo not interfere with binding of the protein to its target molecule canbe derivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of a kinase-binding proteinand a candidate compound are incubated in the kinase protein-presentingwells and the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the kinase protein targetmolecule, or which are reactive with kinase protein and compete with thetarget molecule, as well as enzyme-linked assays which rely on detectingan enzymatic activity associated with the target molecule.

Agents that modulate one of the kinases of the present invention can beidentified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of kinase protein activity identified according to these drugscreening assays can be used to treat a subject with a disorder mediatedby the kinase pathway, by treating cells or tissues that express thekinase. Experimental data as provided in FIG. 1 indicates expression inhumans in uterus endometrium adenocarcinoma, testis, lung fibroblasts,kidney renal cell adenocarcinoma, and the brain. These methods oftreatment include the steps of administering a modulator of kinaseactivity in a pharmaceutical composition to a subject in need of suchtreatment, the modulator being identified as described herein.

In yet another aspect of the invention, the kinase proteins can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the kinase and are involved in kinase activity.Such kinase-binding proteins are also likely to be involved in thepropagation of signals by the kinase proteins or kinase targets as, forexample, downstream elements of a kinase-mediated signaling pathway.Alternatively, such kinase-binding proteins are likely to be kinaseinhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a kinase proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming akinase-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the kinase protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a kinase-modulating agent, an antisense kinasenucleic acid molecule, a kinase-specific antibody, or a kinase-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

The kinase proteins of the present invention are also useful to providea target for diagnosing a disease or predisposition to disease mediatedby the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in uterus endometrium adenocarcinoma,testis, lung fibroblasts, kidney renal cell adenocarcinoma, and thebrain. The method involves contacting a biological sample with acompound capable of interacting with the kinase protein such that theinteraction can be detected. Such an assay can be provided in a singledetection format or a multi-detection format such as an antibody chiparray.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered kinase activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the kinase protein in which one ormore of the kinase functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and kinase activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in humansin uterus endometrium adenocarcinoma, testis, lung fibroblasts, kidneyrenal cell adenocarcinoma, and the brain. Accordingly, methods fortreatment include the use of the kinase protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe kinase proteins. Antibodies can be prepared from any region of thepeptide as described herein. However, preferred regions will includethose involved in function/activity and/or kinase/binding partnerinteraction. FIG. 2 can be used to identify particularly importantregions while sequence alignment can be used to identify conserved andunique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates that thekinase proteins of the present invention are expressed in humans inuterus endometrium adenocarcinoma, testis, lung fibroblasts, and kidneyrenal cell adenocarcinoma, as indicated by virtual northern blotanalysis. In addition, tissue-screening panels indicate expression inthe brain. Further, such antibodies can be used to detect protein insitu, in vitro, or in a cell lysate or supernatant in order to evaluatethe abundance and pattern of expression. Also, such antibodies can beused to assess abnormal tissue distribution or abnormal expressionduring development or progression of a biological condition. Antibodydetection of circulating fragments of the full length protein can beused to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in uterus endometrium adenocarcinoma, testis, lungfibroblasts, kidney renal cell adenocarcinoma, and the brain. If adisorder is characterized by a specific mutation in the protein,antibodies specific for this mutant protein can be used to assay for thepresence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin uterus endometrium adenocarcinoma, testis, lung fibroblasts, kidneyrenal cell adenocarcinoma, and the brain. The diagnostic uses can beapplied, not only in genetic testing, but also in monitoring a treatmentmodality. Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in humans in uterus endometriumadenocarcinoma, testis, lung fibroblasts, kidney renal celladenocarcinoma, and the brain. Thus, where a specific protein has beencorrelated with expression in a specific tissue, antibodies that arespecific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the kinase peptide to a binding partnersuch as a substrate. These uses can also be applied in a therapeuticcontext in which treatment involves inhibiting the protein's function.An antibody can be used, for example, to block binding, thus modulating(agonizing or antagonizing) the peptides activity. Antibodies can beprepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornuleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a kinase peptide or protein of the present invention (cDNA,transcript and genomic sequence). Such nucleic acid molecules willconsist of, consist essentially of, or comprise a nucleotide sequencethat encodes one of the kinase peptides of the present invention, anallelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NOS:1and 4, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NOS:2 and 5. A nucleic acid molecule consists of a nucleotidesequence when the nucleotide sequence is the complete nucleotidesequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQID NOS:1 and 4, transcript sequence and SEQ ID NO:3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NOS:2 and 5. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NOS:1 and4, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NOS:2 and 5. A nucleic acid molecule comprises a nucleotide sequencewhen the nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the kinase peptide alone, thesequence encoding the mature peptide and additional coding sequences,such as a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), the sequence encoding the mature peptide, with or without theadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut non-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the nucleic acid moleculemay be fused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the kinase proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in FIGS. 1 and 3. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel kinase protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 2 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data.

FIG. 3 provides information on SNPs that have been found in the geneencoding the kinase protein of the present invention. SNPs wereidentified at 26 different nucleotide positions (SNPs were alsoidentified at an additional 30 nucleotide positions 3′ of the ORF, asprovided in U.S. Ser. No. 60/265,151 filed Jan. 31, 2001). Some of theseSNPs, which are located outside the ORF and in introns, may affectcontrol/regulatory elements.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45 C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2. Asillustrated in FIG. 3, SNPs were identified at 26 different nucleotidepositions (SNPs were also identified at an additional 30 nucleotidepositions 3′ of the ORF, as provided in U.S. Ser. No. 60/265,151,Attrny. Dkt. No. CL1098-PROV, filed Jan. 31, 2001).

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins:

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. The gene encoding the novel kinase protein of thepresent invention is located on a genome component that has been mappedto human chromosome 2 (as indicated in FIG. 3), which is supported bymultiple lines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates that thekinase proteins of the present invention are expressed in humans inuterus endometrium adenocarcinoma, testis, lung fibroblasts, and kidneyrenal cell adenocarcinoma, as indicated by virtual northern blotanalysis. In addition, tissue-screening panels indicate expression inthe brain. Accordingly, the probes can be used to detect the presenceof, or to determine levels of, a specific nucleic acid molecule incells, tissues, and in organisms. The nucleic acid whose level isdetermined can be DNA or RNA. Accordingly, probes corresponding to thepeptides described herein can be used to assess expression and/or genecopy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease inkinase protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a kinase protein, such as by measuring alevel of a kinase-encoding nucleic acid in a sample of cells from asubject e.g., mRNA or genomic DNA, or determining if a kinase gene hasbeen mutated. Experimental data as provided in FIG. 1 indicates that thekinase proteins of the present invention are expressed in humans inuterus endometrium adenocarcinoma, testis, lung fibroblasts, and kidneyrenal cell adenocarcinoma, as indicated by virtual northern blotanalysis. In addition, tissue-screening panels indicate expression inthe brain.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate kinase nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe kinase gene, particularly biological and pathological processes thatare mediated by the kinase in cells and tissues that express it.Experimental data as provided in FIG. 1 indicates expression in humansin uterus endometrium adenocarcinoma, testis, lung fibroblasts, kidneyrenal cell adenocarcinoma, and the brain. The method typically includesassaying the ability of the compound to modulate the expression of thekinase nucleic acid and thus identifying a compound that can be used totreat a disorder characterized by undesired kinase nucleic acidexpression. The assays can be performed in cell-based and cell-freesystems. Cell-based assays include cells naturally expressing the kinasenucleic acid or recombinant cells genetically engineered to expressspecific nucleic acid sequences.

The assay for kinase nucleic acid expression can involve direct assay ofnucleic acid levels, such as mRNA levels, or on collateral compoundsinvolved in the signal pathway. Further, the expression of genes thatare up- or down-regulated in response to the kinase protein signalpathway can also be assayed. In this embodiment the regulatory regionsof these genes can be operably linked to a reporter gene such asluciferase.

Thus, modulators of kinase gene expression can be identified in a methodwherein a cell is contacted with a candidate compound and the expressionof mRNA determined. The level of expression of kinase mRNA in thepresence of the candidate compound is compared to the level ofexpression of kinase mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate kinase nucleic acid expression in cells andtissues that express the kinase. Experimental data as provided in FIG. 1indicates that the kinase proteins of the present invention areexpressed in humans in uterus endometrium adenocarcinoma, testis, lungfibroblasts, and kidney renal cell adenocarcinoma, as indicated byvirtual northern blot analysis. In addition, tissue-screening panelsindicate expression in the brain. Modulation includes both up-regulation(i.e. activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

Alternatively, a modulator for kinase nucleic acid expression can be asmall molecule or drug identified using the screening assays describedherein as long as the drug or small molecule inhibits the kinase nucleicacid expression in the cells and tissues that express the protein.Experimental data as provided in FIG. 1 indicates expression in humansin uterus endometrium adenocarcinoma, testis, lung fibroblasts, kidneyrenal cell adenocarcinoma, and the brain.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe kinase gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in kinase nucleic acid expression, and particularlyin qualitative changes that lead to pathology. The nucleic acidmolecules can be used to detect mutations in kinase genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the kinase gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the kinase gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a kinase protein.

Individuals carrying mutations in the kinase gene can be detected at thenucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the kinaseprotein of the present invention. SNPs were identified at 26 differentnucleotide positions (SNPs were also identified at an additional 30nucleotide positions 3′ of the ORF, as provided in U.S. Ser. No.60/265,151 filed Jan. 31, 2001). Some of these SNPs, which are locatedoutside the ORF and in introns, may affect control/regulatory elements.The gene encoding the novel kinase protein of the present invention islocated on a genome component that has been mapped to human chromosome 2(as indicated in FIG. 3), which is supported by multiple lines ofevidence, such as STS and BAC map data. Genomic DNA can be analyzeddirectly or can be amplified by using PCR prior to analysis. RNA or cDNAcan be used in the same way. In some uses, detection of the mutationinvolves the use of a probe/primer in a polymerase chain reaction (PCR)(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCRor RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see,e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa etal., PNAS 91:360-364 (1994)), the latter of which can be particularlyuseful for detecting point mutations in the gene (see Abravaya et al.,Nucleic Acids Res. 23:675-682 (1995)) This method can include the stepsof collecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

Alternatively, mutations in a kinase gene can be directly identified,for example, by alterations in restriction enzyme digestion patternsdetermined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantkinase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the kinase gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment. FIG. 3 provides information on SNPs that have been foundin the gene encoding the kinase protein of the present invention. SNPswere identified at 26 different nucleotide positions (SNPs were alsoidentified at an additional 30 nucleotide positions 3′ of the ORF, asprovided in U.S. Ser. No. 60/265,151 filed Jan. 31, 2001). Some of theseSNPs, which are located outside the ORF and in introns, may affectcontrol/regulatory elements.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol kinase gene expression in cells, tissues, and organisms. A DNAantisense nucleic acid molecule is designed to be complementary to aregion of the gene involved in transcription, preventing transcriptionand hence production of kinase protein. An antisense RNA or DNA nucleicacid molecule would hybridize to the mRNA and thus block translation ofmRNA into kinase protein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of kinase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired kinase nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the kinase protein, such as substratebinding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in kinase gene expression.Thus, recombinant cells, which include the patient's cells that havebeen engineered ex vivo and returned to the patient, are introduced intoan individual where the cells produce the desired kinase protein totreat the individual.

The invention also encompasses kits for detecting the presence of akinase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the kinase proteins of the presentinvention are expressed in humans in uterus endometrium adenocarcinoma,testis, lung fibroblasts, and kidney renal cell adenocarcinoma, asindicated by virtual northern blot analysis. In addition,tissue-screening panels indicate expression in the brain. For example,the kit can comprise reagents such as a labeled or labelable nucleicacid or agent capable of detecting kinase nucleic acid in a biologicalsample; means for determining the amount of kinase nucleic acid in thesample; and means for comparing the amount of kinase nucleic acid in thesample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect kinase protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIGS. 1 and 3 (SEQ ID NOS: 1, 3, and4).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application W095/11995 (Chee et al.),Lockhart, D. J. et al (1996; Nat. Biotech. 14: 1675-1680) and Schena, M.et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which areincorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application W095/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the kinase proteins/peptides of the present invention.In detail, such methods comprise incubating a test sample with one ormore nucleic acid molecules and assaying for binding of the nucleic acidmolecule with components within the test sample. Such assays willtypically involve arrays comprising many genes, at least one of which isa gene of the present invention and or alleles of the kinase gene of thepresent invention. FIG. 3 provides information on SNPs that have beenfound in the gene encoding the kinase protein of the present invention.SNPs were identified at 26 different nucleotide positions (SNPs werealso identified at an additional 30 nucleotide positions 3′ of the ORF,as provided in U.S. Ser. No. 60/265,151 filed Jan. 31, 2001). Some ofthese SNPs, which are located outside the ORF and in introns, may affectcontrol/regulatory elements.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified kinase gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

Vectors/host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSecl (Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such as kinases,appropriate secretion signals are incorporated into the vector. Thesignal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with kinases, the protein can be isolated from the host cell bystandard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. The peptidecan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, anion orcationic exchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a kinaseprotein or peptide that can be further purified to produce desiredamounts of kinase protein or fragments. Thus, host cells containingexpression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe kinase protein or kinase protein fragments, such as those describedabove as well as other formats known in the art. Thus, a recombinanthost cell expressing a native kinase protein is useful for assayingcompounds that stimulate or inhibit kinase protein function.

Host cells are also useful for identifying kinase protein mutants inwhich these functions are affected. If the mutants naturally occur andgive rise to a pathology, host cells containing the mutations are usefulto assay compounds that have a desired effect on the mutant kinaseprotein (for example, stimulating or inhibiting function) which may notbe indicated by their effect on the native kinase protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a kinase proteinand identifying and evaluating modulators of kinase protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the kinase proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the kinase protein to particularcells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing. recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, kinase protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo kinaseprotein function, including substrate interaction, the effect ofspecific mutant kinase proteins on kinase protein function and substrateinteraction, and the effect of chimeric kinase proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more kinase proteinfunctions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 10 <210> SEQ ID NO 1 <211> LENGTH: 2203<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1gtgagtcata tgaaagctcc acgctgctga cctctggcaa aaagggagag aa#caaggata     60ggagaggcag tgggggaaag gttcaagtgc gggttttctc cttgaaccta ga#agattatg    120ggtcaagagc tgtgtgcaaa gactgtacag cctggatgca gctgctacca tt#gttcagag    180ggaggcgagg cacacagctg tcggaggagt cagcctgaga ccacggaggc tg#cgttcaag    240ctaacagacc taaaagaagc atcatgttcc atgacttcat ttcaccccag gg#gacttcaa    300gctgcccgtg cccagaagtt caagagtaaa aggccacgga gtaacagtga tt#gttttcag    360gaagaggatc tgaggcaggg ttttcagtgg aggaagagcc tcccttttgg gg#cagcctca    420tcttacttga acttggagaa gctgggtgaa ggctcttatg cgacagttta ca#aggggatt    480agcagaataa atggacaact agtggcttta aaagtcatca gcatgaatgc ag#aggaagga    540gtcccattta cagctatccg agaagcttct ctcctgaagg gtttgaaaca tg#ccaatatt    600gtgctcctgc atgacataat ccacaccaaa gagacactga cattcgtttt tg#aatacatg    660cacacagacc tggcccagta tatgtctcag catccaggag ggcttcatcc tc#ataatgtc    720agacttttca tgtttcaact tttgcggggc ctggcgtaca tccaccacca ac#acgttctt    780cacagggacc tgaaacctca gaacttactc atcagtcacc tgggagagct ca#aactggct    840gattttggtc ttgcccgggc caagtccatt cccagccaga catactcttc ag#aagtcgtg    900accctctggt accggccccc tgatgctttg ctgggagcca ctgaatattc ct#ctgagctg    960gacatatggg gtgcaggctg catctttatt gaaatgttcc agggtcaacc tt#tgtttcct   1020ggggtttcca acatccttga acagctggag aaaatctggg aggtgctggg ag#tccctaca   1080gaggatactt ggccgggagt ctccaagcta cctaactaca atccaggtaa ta#ttgatctg   1140agcttttgaa tactctgaga attagtaatg taaggagagc attggccacg ct#aacagggc   1200gttcttgtat tgtgaactca gcggcaaaga tgggtgtaga ggaatttcta ca#ttcatata   1260ttccctgact aatctttgta tgaggaagac actgaaagag tagctgaggt ta#gaccagtt   1320ccccagctct gtaaaacaca agtagcaagc tgaatagaat ttgaaatgac ta#ttactgtg   1380gattccacat ccattgtcaa atacccaatg gctcaaaaga acaactcaaa ag#atgggctc   1440acttttgggc cccctgactg tcataagtgt attgattagt attgaattgc at#atgtataa   1500aaagaaagct aatgcaacag aacagaggta gaggctcgct aggcctagga ca#tgccaagt   1560aagctgtctg taggttatac ttactaagag ttcattcatt gcctgtaaac ct#gacacttg   1620gtcattgtct ctcacacatt tcatctttca agactggctt ctgggatcga tt#tagaagtg   1680ctggaagtgt tatccatggg ggaattcttt gagaagctgt cgcagggcca ca#tcagaggg   1740atcagattaa gcagtagtca cttcaaggat gttgagacag aggggaggag ac#aggcactg   1800aactacagga tgaaggatca tattagaagc tgaagaagca aataaagccc at#gccaaagc   1860tgagctctca ctggcagggt tgaaggggag gtagaaaggt acagataacg ac#aagattag   1920ggtggatatg ctccaagcca gatttttcta gtctttatgg tcttacattg tt#ccattact   1980aaaaatgaaa tcttcccaaa ttgttgtcct tacttttttt tttttttttt ga#gatggagt   2040tttgctctta tcgcccaggc tggagtgcag tgagccgaga ttgcgccact gc#atgtccgc   2100agtccgacct gggcgacaga gcgagactcc gtctcaaaac taaaaaaaaa aa#aaaaaaaa   2160 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa    #                 220 #3 <210> SEQ ID NO 2 <211> LENGTH: 343<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2Met Gly Gln Glu Leu Cys Ala Lys Thr Val Gl #n Pro Gly Cys Ser Cys 1               5   #                10   #                15Tyr His Cys Ser Glu Gly Gly Glu Ala His Se #r Cys Arg Arg Ser Gln            20       #            25       #            30Pro Glu Thr Thr Glu Ala Ala Phe Lys Leu Th #r Asp Leu Lys Glu Ala        35           #        40           #        45Ser Cys Ser Met Thr Ser Phe His Pro Arg Gl #y Leu Gln Ala Ala Arg    50               #    55               #    60Ala Gln Lys Phe Lys Ser Lys Arg Pro Arg Se #r Asn Ser Asp Cys Phe65                   #70                   #75                   #80Gln Glu Glu Asp Leu Arg Gln Gly Phe Gln Tr #p Arg Lys Ser Leu Pro                85   #                90   #                95Phe Gly Ala Ala Ser Ser Tyr Leu Asn Leu Gl #u Lys Leu Gly Glu Gly            100       #           105       #           110Ser Tyr Ala Thr Val Tyr Lys Gly Ile Ser Ar #g Ile Asn Gly Gln Leu        115           #       120           #       125Val Ala Leu Lys Val Ile Ser Met Asn Ala Gl #u Glu Gly Val Pro Phe    130               #   135               #   140Thr Ala Ile Arg Glu Ala Ser Leu Leu Lys Gl #y Leu Lys His Ala Asn145                 1 #50                 1 #55                 1 #60Ile Val Leu Leu His Asp Ile Ile His Thr Ly #s Glu Thr Leu Thr Phe                165   #               170   #               175Val Phe Glu Tyr Met His Thr Asp Leu Ala Gl #n Tyr Met Ser Gln His            180       #           185       #           190Pro Gly Gly Leu His Pro His Asn Val Arg Le #u Phe Met Phe Gln Leu        195           #       200           #       205Leu Arg Gly Leu Ala Tyr Ile His His Gln Hi #s Val Leu His Arg Asp    210               #   215               #   220Leu Lys Pro Gln Asn Leu Leu Ile Ser His Le #u Gly Glu Leu Lys Leu225                 2 #30                 2 #35                 2 #40Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Il #e Pro Ser Gln Thr Tyr                245   #               250   #               255Ser Ser Glu Val Val Thr Leu Trp Tyr Arg Pr #o Pro Asp Ala Leu Leu            260       #           265       #           270Gly Ala Thr Glu Tyr Ser Ser Glu Leu Asp Il #e Trp Gly Ala Gly Cys        275           #       280           #       285Ile Phe Ile Glu Met Phe Gln Gly Gln Pro Le #u Phe Pro Gly Val Ser    290               #   295               #   300Asn Ile Leu Glu Gln Leu Glu Lys Ile Trp Gl #u Val Leu Gly Val Pro305                 3 #10                 3 #15                 3 #20Thr Glu Asp Thr Trp Pro Gly Val Ser Lys Le #u Pro Asn Tyr Asn Pro                325   #               330   #               335Gly Asn Ile Asp Leu Ser Phe             340 <210> SEQ ID NO 3<211> LENGTH: 53332 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 3tataggccaa tgctgtggct cacgcgtgta ttcccagcac tttgggaggc ag#gaggatcg     60ttgagctca ggaattggag acaagcctac gtaacatagt gaaacctctg tct#gtacaaa     120aataaaaga attttccagg catggtggcg tgcaccccca gtgccagcta ttt#gggaggc     180gaggtagga ggaatgcttg aagccaggag ttgaagacaa gcctaggcaa cat#agtgaga     240cctgtgtct ataaaaaata attagctggt tgtcttggca caggcctgca gct#agctact     300ggaagactg aggtgggagg atcactgagc ccaggaggct gaggctgcag tga#acagtga     360cacccagct ggattccagc ctggaagaca gagggagacc ctgtttccaa aaa#aaaaaaa     420aaaaaaaat gcaagaaaag acatcataaa cttgacctgg gacataactt tta#tgtgatg     480aattcacaa tcttttagga agaaattagc atttctgata aaatgtatta taa#ttatatt     540ttataaatt caaatggaat taaatattct gagaaactag cttctcactc tct#cagttgt     600agtcaaaac tttaatggtc tttggccggg tgcggtggct cacgcctgta atc#ccagcac     660ttgggaggc cgaggcgggt ggatcacaag gttaggagat cgagaccatc ctg#gctaaca     720ggtgaaacc tcgtctctac taaaaataca aaaaattagc cgggtgcggt gcc#agacgcc     780gtagtccca gctgctcagg aggctgaggc aggagaatgg tgtgaacccg gga#ggcggag     840ttgcagtga gccgagattg cgccactgca ctccagcctg ggcgacagtg cga#gactctg     900ctcaaaaaa aaaaaaaaaa aaaagttgaa tggtctttga gccaagtagt ctt#ccttttc     960ttcttcttct tttttttttt ttttcaaaaa atatctctag attgaatctt gg#aattggct   1020taagtctctt ctcttgtggc aattttgaaa tgaaaaaata catgctcata at#taaattac   1080ctgaacattt taaaaaacca tcatgaggtt caaatatcaa atattcataa at#attgttgt   1140gataatagac ataactctta ttttttccct taataatgat tgtttatata tc#ctccattc   1200tgtctcactt tatgattagt atattatagt ggcaataatc ttaggaatct aa#cagagaaa   1260agtgttgcat ttgaagacta cagactgcaa accaatttaa gccagattcc tt#gacatgtt   1320gtgctgttaa tatagtactt tacatatagt aaacattaat tacatatatg tg#gaaggaag   1380caagcaagaa aggaagaaag tatttcattc aaactcctct ctctccatca cc#attggcta   1440atatcatcat ttgtacagtt aagaacaaca taggtgctca ccacatagtt tt#tgaataaa   1500tgaatgaatg gcaacccttc taagactatt ggatacacta ttgtttgaag gc#aaagagat   1560gcagtagata ttttcaactt ttttcctgtt ttatgattct gtggtttctt tg#actactaa   1620aagttagcta ggtagcaaat ttgttttaaa gtctgaaaac caaaatgctt tc#agataaaa   1680ggtagggaga aaaatactcc tcaacatgtc cactttagca ccaggaaaac ct#aatatcaa   1740tatcaccatc aatgatatca tataaatatc attgcataga taagcaatgt ca#atccctaa   1800aaactatgta taccaatagc actaacttgt ggccagaaca agaaccttaa ct#gtgccaaa   1860ttttattcta ttcaataaca gctgcctcgt tttcagttgt gcacatctga at#gcaagcaa   1920tccctgtctg atgtggagtt tcttgcactg ataaggaaaa actgctgaag tt#gtgaggct   1980gctccaggca gagccatcat gtgagtcata tgaaagctcc acgctgctga cc#tctggcaa   2040aaagggagag aacaaggata ggagaggcag tgggggaaag gttcaagtgc gg#gttttctc   2100cttgaaccta caagattatg ggtcaagagc tgtgtgcaaa gactgtacag cc#tggatgca   2160gctgctacca ttgttcagag ggaggcgagg cacacagctg tcggaggagt ca#gcctgaga   2220ccacggaggc tgcgttcaag gtatttgtat cccaggagag agcatctttc tc#tattgata   2280aaccaaggag ttcagacact ccctttttgt agcgggatct gattcttctg cg#gtaggtct   2340aaaccaataa aatgaaaatt ctattaaagt cacagaaaat ttatggctgt ag#ttatcaaa   2400tttggggaat ttcttgtaaa ccaaaaggga aaaataatcc ttggctttgg gc#tgcacgaa   2460actcacttgg cttgaagtcg agaaagtagt tctctcaaaa tctctaaggt cc#taaattac   2520agagctgaaa cttaaaaggc aagctgcagt attagttggt atgctatgga tt#tgaaactt   2580tagtaattag ttcatgatta ttagcaatgc catagattat tcccctacag ca#ataaatta   2640agtggacatg aaaaaaaaaa gccagactta aacagaaaaa agttgcaaaa ca#tccatcaa   2700agagatttag gttaacctga atgttaaaga cacattttta ggtgaagaaa ga#atgtagta   2760tttcaggagt tgataccatt atggtctttt tcagggatct ttcaagaaaa gt#gccttttg   2820ggggtacagg aagcttagaa aacatttgaa gagtgaaaat gaggcaaata aa#gaaaaaat   2880ggttttacca ggcactgaat ctttactttg cataaatttt atttctgctc tt#tctttttt   2940ctctagctaa cagacctaaa agaagcatca tgttccatga cttcatttca cc#ccagggga   3000cttcaagctg cccgtgccca gaagttcaag agtaaaaggc cacggagtaa ca#gtgattgt   3060tttcaggaag aggatctgag gcagggtttt cagtgggtga gtgagcagct ga#tgttgatc   3120aagaagaatt taatgtgagc ttgtctacgg aggccggccc ttgcttccag gg#caattact   3180gagcgagcct tcccaagtct gctctggcaa tgctgtctaa tttccctggg ga#aaaaaagt   3240caacactaaa aaaaagtgtt ctttctctct tccctttcac ccgctccttt tc#cccattcc   3300cctagagcag aggaagagcc tcccttttgg ggcagcctca tcttacttga ac#ttggagaa   3360gctgggtgaa ggctcttatg cgacagttta caaggggatt agcaggtgag tg#acacatag   3420ctgggagaga ctttagagat gagagtcccg cccccccaat ttcatattat aa#agccaggt   3480gagacatcat agaagttcat agcactcagg acctgtgcaa gacaccatgg cc#gacaggga   3540gagagacatg ataacttaaa cagccttgaa agaaaaacaa acctgccctg cc#ctaattaa   3600aatcagccca cttaaatgtt tatcagcctt tcccttcttg cattcaattc ag#agaattca   3660aagaaaatag acattctcta ctactgaccc aaagaacaat tatcactctt ca#ggcctgtg   3720ggaggcacag ttggtaaagc gtctctaaca ggttttttat atccctccct aa#atcacaat   3780gacagagttt tgtaatggca acctggaatt tgctgcttca ttcctccacc tg#gcctttat   3840agaagaaact gaagttggtt tctgcaaatt atggtacatg caaaagatga ta#aatcctag   3900attttttata tttgcaaaat acacaaaatg tctggagaat aaaaatactg ct#tatccaaa   3960agctaagtac taattttggt aaacaaccaa ctttgttaaa tatatgtaaa ag#atccatga   4020attccccttt tagtcaaggt gggaaagttg gatggtcgct tttttcttta tg#ttactcca   4080atagagagaa aagtaatggc tcaatagtgg ttaaatatta attttaaaaa ta#tagctgat   4140ccgagtgcag tggtgtttac aactacttga tcacaaccag ttacagattt ct#ttgttcct   4200tctccactcc cactgcttca cttaactggc caaaaacgaa aaaagaaaaa tt#ttatataa   4260ctactacaag actaaatatt tattatttat cttagtattt atgctgttat ta#ttattttt   4320acttgttaaa acaggattgt aggggacata cagttttatt ttattttatt at#ttatatat   4380ttatttattt attttggaat ggaatctctg tcacccacgc tggagtgcag tg#gtgcgatc   4440tcagatgact gcaacctctg cctcctgagt tcaagcaact ctcctgcccc tg#gcccttta   4500tactttctta atctgtttta gtcatggtgt accttaactt ttttcaatgc tg#agaacatc   4560tgcaataaag gaccacattt tattttattc taagcttcct catatcaatt tg#gccatggt   4620aactgttttc aaggtggctc ggaacggggg caccctggaa catacttgga ta#catgggca   4680ccatggacac ttctgatcct ctcttctgag ttctgacttt gattgttctg ca#cagacctt   4740tccagcccga agtttacaca gaattcactt atcttttctt ctagttactt ta#tgttttct   4800ttttcattta actctttcat ctactgggaa tttatattgt atattcacaa tc#accccagc   4860tccatttatt agattttctt ttctctgatg gtttgaaatg ctgccatgat ta#tatattag   4920atctcacgaa tacttgaaat tctttctgtt ctaatctttt aaaaatcatg tt#tccttaat   4980ctatcttttc ttatatttgt gctgcatgat tttaattatt gttgctttag gc#tattttta   5040gaatatatca aaactctacg ttagagaatt attgacatct ttgcattatt ag#attttcta   5100atacaaatat cctgtaaata tctaatacaa cagtctctgg atggtcactg ta#caagaccc   5160tatagaatcc ctaccctcca ttccccggca cacactcagc tcctccctgt cc#tcatctcc   5220ttcccctctc ctgcttcaat gacagactgc tcctgcctca gtcaaggact tt#taacttgc   5280tgttccctct gcctggagct gccttccact gttcatgcac acagctgact cc#ccctcgcc   5340atcagattcc tggttcaagt gttaccttat ttataaaact gtagtcccag ct#agtccagg   5400gaggctggag gcaggagaat cacttgaact ttggaggcag aggttgcagt ga#gctgagat   5460cggcaccacc gcactccagc ctgggtgaga gtgacactgt ctcaaaaaaa aa#aaaaagca   5520ttttctctta taaacatatt tgccaaaaaa ctttttgcag ggtttggggg ag#aatttcac   5580agaaccatgt tctgaggaaa atacttacct cataaaactc taaaacaaaa tt#tcaaagac   5640atgataaggc aaacaaaaga aactggggaa aagtatatgc aaaatagttc aa#taaaaagg   5700tgggcaaatc ggcaaatcac aagaaaaaca gaaaagatcc ataaacttat ga#aaagtcag   5760tttcacatat ggttaaagaa atataaatta aaatgcgata aaccttttta ct#tttcaaat   5820aggccaaaaa aaaaaagaag atgaaagcga aaagccaacc cacatgatag gg#ctatgaca   5880gagggacaca ggagccaact gaaagagctt ccaaaggaca aagctgcaaa aa#tatgagca   5940accaaaaaaa gtggtattaa attataaccc aaagtataaa ataaatatct at#gagtccgt   6000actgatataa ataaatgatt caatacatta acaaatggga gagaagaaac aa#atctctca   6060tgccaaataa atacaaataa tttatgtaga taatatacct tcaaagaggt ac#agcataac   6120tctccactcc ttaagtgtgg gtcattcata gtggcatttc tctaaaagta ca#gtatgaaa   6180aagggggaga aagagtaact ttagagtaga gaaacctgac caacactatc tc#agacaggt   6240gactaaggtc aacatcaaaa gtcataaatc atgatgatgg tatgcactct tt#tttttttt   6300tttttttttt ttctcagatg gagtctcact ctgtcgccca ggctggggtg ca#gtggcgca   6360atctcagctc actgcaacct ccggctcccg ggttcaagcg attctcctct ca#gcctcctg   6420agtagctggg atcacaggcg cgtgccacca tacccggcta attttttgta tt#ttagtaga   6480gacggggttt caccatgttg cccaggctgg tctcaaactc ccgagctcag gc#aatccacc   6540cacctcaacc tcccaaagtg ctaggattac aggcatgagc cactgcgcct gg#ctgagggt   6600atgcactttt tttttttttg agacggagtc ttgctctgtc gcccaggctg ga#gtgcagtg   6660gcacgatctt ggctcactgc aagctccgcc tcccaggttc acgccattct cc#tgcctcag   6720cctccccagt agctgggact acaaggtgcc ccaccaccca cacccggcta at#tttttgta   6780tttttagtag agacggggtt tcactgtgtt aggcaggatg gtctcgatct cc#tgacctcc   6840tgatccaccg gccttcgcct cccaaagtgc tgggattaca ggcgtgagcc ac#tgtgcccg   6900gcctgatgaa atgttaaatc tttattaaat atcggattgt acaagaatga ac#tataagag   6960aaaagttaca tggaggaaaa aaggttacta acaatatgat tttaatccca ct#gtattaaa   7020aacaatggat ttatacctgc attaaaatct tctctattct cagcacttag ct#gatatgaa   7080taaaatgatg aatgagggga cagtaggagg aaatgaagag agagagaata at#ggtgtggc   7140ctgggaagat caggtagcac ttagaagccc gctgcaagaa tttggctttt at#tctaagta   7200atgcgtggag atatggtggc ttttgaacag aaaagtgact tgtcctgatt gt#catttgaa   7260aagtatgcct ccaactacta ctgctgagag taaatagtag gagtgcaagt gt#gctcagca   7320gggaaactgt tagaagacca ctacaaggct gggcttggtg gctcgtgcct gt#aatcccag   7380cactttggga gcctgacgtg ggcagatcac ctgaggtcag gagttcgaga cc#agcctggc   7440caaaatggtg aaacccccat ctctgctaaa aatacaaaaa ttagccaggt gt#ggtggggg   7500tcccctgtaa tcccagcttc ttgggaggct gaggcaggag aattgcttga ac#ccaggagg   7560tggaggttgc agtgagccaa gatcgtgcca ctgtactcca gcctgggcaa ca#gagcgaga   7620ttctgtctca aaaaaaaaaa aaaaaaacaa aaaaacaaaa aaacactaca at#aagtcaga   7680tgaaaaataa taataagctc caaattttct ataatggaca tatatatata ta#tcacttta   7740gtaaagaggg aaaatgcttt ggaatatata tgttatatat gtattgatac at#gttaaact   7800ttttattttg agaaaattat agatttatat gctagaatat attttgaagt ga#aagtgctt   7860ttgttaagcc atctttggta taaattgctg ctttgaacca cctcaataag tg#tgtgcccc   7920tcaatccctc tcttctagaa taaatggaca actagtggct ttaaaagtca tc#agcatgaa   7980tgcagaggaa ggagtcccat ttacagctat ccgagaaggt aagaacagca ga#aatggacc   8040caatagatct gttttgagtc cttgatttgg taaaaaatgt attgcattga tc#cattcagc   8100atctagtttt gattcttctg gaatactata attacatttt tatttttcat ac#aagttttt   8160caagaaattt acactgctat tttattactt aattttgagg aaattgagat tt#aaaactat   8220tatatcactt gaccaaaact ataaattcac tgagcaatta ctaatacttt cc#atgtgttt   8280ggcctcatgc taggtgctaa ggctatacct atataacctc agaaaattcc ta#taaaagag   8340aaaatatata atcacacaaa ttcttactgg gaaatttgcc tgaacataac at#gttgttag   8400ctagcacttg gagattctcc agaaggcatg catgtttagt gttactgcct gt#attttctc   8460tgtgccctgg acagtacagc aaatgggtga ggaacctggt gtcaaatgga ct#tgggtttg   8520cagcacaggt ccaccaatca ctagtggtat gatgttgggt aggttacttt ag#ctatttat   8580tactcagttt cttgcaggaa gaggataata gtggtaccta tttcatggag tt#gttatgag   8640tattcaacaa gaatatgtat ataaagcact tatcacagag tcagtttttc ag#agttcaac   8700aaatgttgac catttttatt ccattcttct tttcctgggt aatgtcttat tt#accatcaa   8760gataactaat actttataac ataaacatca agaagccaac atagtgaaat ga#atcattaa   8820aaatataatt tatcaacctt tattgcatga gccatttgaa ataagatgat ga#taggattg   8880ctatgcattt cagcaaaatc ccagagaaat ggcacttccc tggccttatt tt#ctcccact   8940tttaactact tatcttctgt tctttactga gcacatgcta tatgcagagt at#gctgctgg   9000atgctgtgaa ggatgagaag agaaacccat gtctttgttc tatcatttgc ag#tcttaaca   9060gagcacatga ttcaagttac aagtgtataa aagacataaa ctaagatgag ag#caagttag   9120tctcagtgtg actgatggag tcactagatt ttgaactgag cttggaagga ta#ggttatgc   9180aaacaagcat ggaaaaagca attcagaaaa tgagtttata actgaatttg at#accctttt   9240caaaagtctt tcagagcccc tgaggaatac atcattttga atttaattgg aa#gggccaaa   9300tgggctattg gtttagccag agattcatcc tggtaggatc aggtgcattc tg#ggagaagg   9360catggtttta agtgtttaat ataatggaaa ctgcattaac taatgtactt at#taatggtc   9420tccatgaaag gatgatcaga tttggaaaga gatgtatgga taggttaaag ag#tatttgtg   9480aacgtaatag aaattcccag gtcacccgca taagaggaag gtttcctttg tg#agcttgag   9540tttgccaatt gcttaagatt ggctttgctt agatattgcc cacagccaag tt#tttcaggt   9600tgacatttaa ctgtaacagt gaaacctttt gccaggtttg ctaacagatg gt#tctcagca   9660tggttcagaa aacctggatc cgttttcttc tgtatgctaa atgtttcttt ca#ttgcatat   9720ttacggagga attgcctctc catcacaggt gtttacaatt acatttagta gt#caactgtg   9780gactttcttg gtttgtttta tggacttacc ttaccgaatg ctttgctcgt gt#aatattaa   9840aaaccacaag aggatttctg acacattgga ggttgttagg aatccaattt cc#aacaatga   9900atgtttcttt ttacaccact ataaaagctt ggagcccttg ttaaaagagc cc#tctcccct   9960caagaagata tgaggcttta ttcgaaaact ttggcactgt cccatttttc ct#gtaagaac  10020tttaaggatg tgagaccagg gagacaggag gttaaatgag aagggctgga ag#gcaaagta  10080agaacagctg gagttcatta gctaaaatcc agggtcacta gctaaaaagg ca#accgaaag  10140gcacgtgcag gaaaactgaa caagtaatgc agccctcttt aaaaagcctt ga#agcaggaa  10200ttgcttttcc tgaacaattt ggctgccctg atggtatagc agccaaagat tt#attaagta  10260tgattttact acatatatgg tctctttcta tacaggtaga atacatgtgg ca#atttacta  10320gtctggtcat ttggagtact attttcattt gaccttaaca tgtgatatta tg#aaactagc  10380aaaagtatga acagcactaa ggaacatttt tttttttttt ttttgagacg aa#gttttgct  10440cttgttgccc aggctggagt gcaatggcac aatcttggct tactgcaacc tc#tgccttcg  10500gggttcaagc aattctcctg cctcagcctc cggagtagct gggattacag gc#atgtgcca  10560ccacacccag ctaattttgt atttttagta gagacagggt ttccccatgt tg#gccaggct  10620ggtcttgaac tcctgacctc aagtgatctg cgtgtctcag cctcccaagg ga#aatatatc  10680ttaatacatg tgtcagtgct tttcatactt ctttcaatcc tcttaacaat ct#ttagagat  10740agatattatt aatattattc cactatatgg tggtgattca aaccaaatct ct#ctgattca  10800aaaattcata ggctttctac gcacccactg tagaaatatt catttagcac ct#actatgac  10860caggtactct gccgaactgc tagatacaca gcaatacaca aaatagatgt gt#tccctacc  10920accctcattc ctttgctaat taagaaaagc agaggccttc atagtgcctt gg#aaatctct  10980cataattgac tctagaattg tattttaagt gttgattttt acaactagga gg#aaatactt  11040tcatttgaat aggctaatgt gttatgtttt tacatagtac aacatttctt ag#ttttatga  11100aactttatag caatatctta atataatgtg cattgtttta aatatttttg tt#caagtggt  11160caacttttgg tttaaactga ggactttcag cctgttaata gcatttttct ta#ggaaggag  11220tcatataact aatctttttt gaggacaagg catatgacat aatctccccc tt#cccctaca  11280taatgtatat ttttaaaacc tttataccaa ccctaggaag taaaatgtgc ta#tttttgtt  11340gtagagataa agaaattcta gcctcagaga ggttagttaa cttgtctgag gt#cacagaga  11400tagtaatcag agttgttaga atccatttct attctattta aaatcccttc ta#ctttatta  11460tgatgaattt ggaaatgctt aactaaagta tttattgttt agcaacagta aa#aataaaaa  11520tagaaatctg tttttattat acattttata taaacgttaa ggaaaatgca ga#agaagtat  11580ttttttaatc tttaatttta gattcaaggg gtacatgtcc aggtttgtta ca#tgagtata  11640ttgcatgatg ctgaggtatc ttgtcaccca aatagtgagt atagtacctg at#aggtagtt  11700tttcaacccg tgtccctctc ccttcctctc cccttttgga gtccctggtg ta#gtgtctat  11760tattcccatc ttatgtctgt gtgttcccaa tacccccagt tattagcttt ca#cttgtaag  11820tgagaacatg tggtatttgt tttctgttcc tgggttaatt cacttaggat aa#tggcctcc  11880atctgcatcc atgttgctgc taaggaaatg gttttttttt tttttttttt tt#gtggctgc  11940atagtgtttt atggtgccag tgtacaaatt ttctttatcc aatccaccat tg#ctgggcac  12000ctaggttgag tccatgtctt tgctattgtg aatagtgctg tgacgaacat aa#aagtctag  12060gtgtcttttt gacagaacga tttattttcc tttgggtata tacccaggaa tg#gaattgct  12120gggtcaaatg gtaattctgt ttttggtttt tttgaggcag gagatgggac tc#gactccag  12180agatggggct tgaacactaa accaaattta ggactagcca aaacagggcc tg#gggggagg  12240cagctttcca gaagacacac ccaccagtgt gccatgtcag tttaccattg cc#atggcaac  12300acctgaaagt taccaccctt tcccgtagca acaacctgac aacctggaat ta#ccactctt  12360ttcctaaaac tttctgcata aactgcccct taatttgcat ataactaaaa gt#gggtataa  12420atataactgt agagctacct atgagctgct actctgggca cactgcctat gt#ggcagccc  12480tgctctgcaa ggagaggtac acccgctgct gctgaacact gctgcttcaa ta#aaagctgc  12540tgtctaacac cacaggctca cccttgaatt ctttcctggg tgaagccaag aa#ccctccca  12600ggctaagccc cagttttggg acttgcctgc cctgcctcac tttgagaaat tt#ctaaactg  12660ttttccacag tggctgaact aattaacatt cccacccaca gtgtataagc ac#tccctttt  12720cttctcaagc ttaccagcat ccattaactt tttacttcta aataatagcc tt#tttgactg  12780gtgtgagatg gtatctcatt gaggttttga tttgcatttc tctgatgatt cg#tgatgttg  12840agcaattttt tcatatgttt gttggccact tgtgtgtcca aaagaaatat tt#taaagaaa  12900ataatacatc atgttgtata ttcatcaatt ctgattctat cattgattct ac#agtgccgg  12960taattgcagt gtttaaatta gaaacagtct cagctaagaa tcttttaaga tc#attctcta  13020gtagaaaaac attacaaagt aatgattccc aatccatata tgagaaaact ga#gccaaaaa  13080taggctaagg agcctcccta aggtcataca atgaggcagg ggaggaggct ga#ttagaact  13140tctgaattgc caatgaccac aaatagtcta gggtaggcct ggttgacaga aa#gtctgcca  13200ttgaacacca tcatatcaca tgacaaatac agcaaattca ttgtgcatag tt#acgtcttt  13260ataaaacaaa ataatgccag gataatggta tgtgatcagc attacaattc ca#aagatacc  13320aagacaacta cttatctgac acttgtctta gtatttctct aacatttatc ta#aaattatt  13380tcaattattt cttttctcgg aatgcataac ttgactcatt gacttgattt at#gattctca  13440gatcaaagga aatgtaacaa cagggactag aaacactttt ttattcaatg tc#caatgagg  13500gttggggagg actccatcat tgactcatta tataattcct cataaactca tt#acaattgg  13560cctggctttc attaattcat gagcacttat tgagcaccac atgccaggcc tg#tgctagtg  13620ctggagatgc aaagacaagg gcaagttcaa tccatgccct caatgagttt ac#agcctaaa  13680gacgactttg actaccaggc cttcattaca tagagcgaca tcctaggact tg#gagaatca  13740gctttcctct ggagccttaa agacatccct atttactttt gtgtcttttc tt#tgaagaaa  13800aacaaaaata agtatacata ggatacatta ataataaaaa aacagtattt ta#tgagactc  13860agaatgctaa ttttaggatc tttgcccttc tcagttgact tttgtgtccc tc#aactgttt  13920agtctgcagg acagatatca catcctgctg tgcagtttat aaaatgtcct ta#aaattaga  13980agaaagaaag gccttgtctt cctgggttta agacccacac atctgaggct gt#aggcattt  14040cagatccctc tggtggatgg accaaaatga taaacaatac tgtgagataa at#gctttaaa  14100catcatctgc tctttcatct gaattcccta ttcattattc ggcaacattc ac#agttttca  14160tataacgatt tcagtagttc tagggcacca gaaaagcagt actaggaatg gc#cataaagc  14220atagaatatt tataatctaa tgagggagac aactaaaaga aagaaggaat aa#aagcatct  14280tcaacagaaa caccctttac caaccaacta gaggtataga aatgatatta gg#taattagt  14340gaccactaat ttaaagataa atatttattg agtgccagac attgttccag gc#actgagta  14400tatagcaata agcaaaaaaa acaaaacaaa acaaaacaaa agtgcccact ct#caatggag  14460tttatattct caattgtgga gacagacaat aaacaaatat ttatatataa aa#tgtcagat  14520ggtggtgaca ggcactatgg aaaagaataa agcagggccc agagagagag gg#taggatgg  14580ggtagaggtg ggatggggtg gagggctgct gaggtgggat ggagtagagg gc#tgctatct  14640cacctagaat ggtcaaggaa gtctgcacct atatgtatca cttgagcgga gg#ctctgaag  14700aaagtgaggg aggatgaagg cagagaggtg agaagagagg attacaggaa aa#gacattgg  14760caagtgtaaa atcctggggt ggaaatgtgt ttgcaagtgt gtctaaggaa ca#gctaggag  14820gccagtgagg ctaaagccaa gtgagcaaag atgggagtgt gaggagatga ca#ggtcacga  14880tgggcacagc caacagtagg gtgggcagga aatcgcaagt cctttgaatt ta#ctctgcag  14940gagatgagag gccactggag ggtttggaac caggaggcac atgccctaac tc#atttgaga  15000aggatagcag tgtctggctg tcctgtgaag aagtggccat aggaggaaag ca#gggaagca  15060ggcatttgca ataattcagc caacatatga tagtggcttg gtccagggtg ct#ggcagaag  15120atatggcaag ggaggggttc tggacaattt ggaaggtaat gccaatagat tt#gtatgtga  15180taaaaagttg agaggacttg acgtgtacga gtggttaatc ttcataaaat gg#atgaatgg  15240ttaaaaagat ttccgcaaag aaactgtggg ttgaaggtaa aactagtaac tc#caatgtaa  15300gtgaacaaca gagaaataca aaacagacat ttttcctact cctacaaaaa ct#gtaattat  15360caagaagacg acatgaagtt tatacccagt attgttagca ggaagcctca tt#ccaagtag  15420atatttttcc ttggccattt tagcaagtga gagcatgagg ccatcataat ga#acaaatca  15480tgccatcatg atttaaaaag aagcatctgg agttttagta atatagttag gt#gagactaa  15540aattatacta aacataaaat taaaatatct taacaatatt cttagcaatt tc#agctttac  15600catatccttt tgaaatctaa ttttgctata tgctttgtaa cataggggtg gg#ggaaagag  15660agaaatttat gagataattt ataaataaaa atacacctaa agtataagca tt#ctcaactg  15720atggtcagaa aatatggaag gtattcaaaa ctctagcaga aacataccat aa#acaagatt  15780ttaagactga aagtagacgt ttagtggggt tcagggtgaa aggcaggggc aa#gaagctgg  15840caagaagagg gaagggatac taattctaat ttgcctctgt aatgctttac at#ttaccaag  15900gttccacaaa tggtatctga ttccatcctc atatcaaccc tatgaagtaa gt#cagaaaag  15960acgatgtctc ttttcctaag gaatgaattg agacttaggt tgagatactc tc#cagagctt  16020actcagatag gaagtgacag ggccaggatt catattaggg cttctggctc ca#cagacagt  16080tctccttaag actttcaata aatatgtttg acaaattaag tgcttactct cg#gctgagtg  16140tggtactagg tggtgtggca gcatctcaaa aagggggaaa gtcactccct ca#attcccat  16200gtggccttca gtctgagact agggagatta aacagatgcc tgagaagctg tt#tattacat  16260ttacaaagca acacatttgt caaagtgaaa taataaattt agcccataag ga#ctctgggg  16320gcaaaaagta aaaattaagg cattagtcat tacagcaaat aaggttaaca gg#tgtgatgg  16380agctccttcg gcgtaagtca gcttaaattg acaagtaaag agagaaattc ac#tggctcac  16440agatctgata actacaggct ggtagggcat aagcaatatc atcaggaagc cg#tgtctctc  16500attacccaac actggtttgc tgtgcattca ttttattccc aggcatgttg tc#accaggtg  16560ttggtaatct gaccccagca actcctggct aaatcccaca ggtttagctc tc#acaataga  16620aaagaaagca cttcttttct aatggcacca gcaaaacagg gtctgccaaa ct#tgggtttt  16680gtgcctgtct ctgaaccaat cactagggta taggggagtg ccgtgctctg at#ggccagcc  16740ctgggtcata tgcccattct tgggtagagg ccgggtcagt tccaccagat ga#gcatggtc  16800tgaggaagaa gacggttgtt tttccagggg aaaatagaag tgcccccgct ag#aagggaga  16860atggctgtca ggagggcaaa acgacagatt cactaaaata ggttgatgcc ta#aagaaaat  16920aattttattc ctaaatttaa gggagtattt cagttgtttt taatcttatg ga#attctaca  16980ctgggaggga gttggtgcag gagattcatg atatgcaggc ataggctaca ga#ataatgct  17040ttgagttttt atcctttact tttcctttcc tttaagcttt aaagacacga tt#tcttcatg  17100cagggttgcc ctgaggtgag cctcatcatc tctttttttt gagatggagt ct#cgctctgt  17160cacccaggcc agagtgcagt ggtgcaatct tggctcactg caacctccac ct#cccaggtt  17220caagtgattc tcttgcctca gcttcccgag tggctgggat tacaggtgtg ca#ccaccagg  17280ccccaccacg cccggctaat ttttgtattt ttagtagaga cggggtttca cc#gcgttggc  17340caggctggtc tcaaactcct gacctcaggt gatccaccca cctcggcctc cc#tgagtgct  17400gggatcacaa gcatgcgcta ccacgcccgg cctcatggtc tctttattgt ac#cttttcta  17460gtctctgctt tcctgaagcc agaggtcttc ctatctccag aagctccaaa ga#cacacttt  17520caaacccctc ccagtcactt ggccttttct gatgacttct ttccttcaag gc#tgccttta  17580gtaaccgatt attgaagagg caagagaaag ccctcagcct tctccacttt ca#cctccctg  17640ggctccccaa gtttggccga ctcctctttt caagttcaca ttttctcctt tc#cacagagg  17700tttgcaacat tacctttaag aaatcatctc cagtctctat cacgtttcaa ca#gttcttta  17760ccccatgctt ttatccctgt ctcccaccaa tcatatccac cggccctatt ga#ccgcttgt  17820gggagttaga attttggaga ctggtcatat gtcacaaagt cctgctctag aa#ggcagaac  17880actccatttc ctgctcctcc aaagcccttt atctctccag gcctctcctc ct#gtagctct  17940gaagctggat tgatgagatt cccagagggg agcatttagt gctctgagtg ct#ttgatgaa  18000attgattagg taaatggaaa catatttttt gcaaccactc tagcctgtag aa#acaataag  18060ttgcaatgat ttgccatttt tgaaataatg aaggttcttt gtaattttaa at#attctttt  18120gccacaagag attgttttcc agcagtaaaa taaccagaat gtttgatttg aa#atgttgaa  18180aaaatatata ccgtctgata tctttagagc agcactttca ttatcaatga tg#gatttaac  18240attttgttta atttttctag cttctctcct gaagggtttg aaacatgcca at#attgtgct  18300cctgcatgac ataatccaca ccaaagagac actgacattc gtttttgaat ac#atggtgag  18360ttgttcgagc attttacaac acttgagaaa aataacctgg tacttgtata at#gaatctgt  18420taatatttta tggcatgata aaacttttat tataatgtga aaagtatcat gg#aaattttc  18480attattgtga ttagtagaac cttattgttc ccacatccat ctttggtcct gc#ttccttac  18540ccatgacttt tgctgtccct tttcccctca tcagcaataa taaatgagga tc#ttgagttt  18600accttctaaa taaaactttt gcacttattt ttaatctaat tttaatcact at#ctgagcag  18660aatccaacat tttttcattg acaataaagg taaaaatcac aagatattta aa#aattgtat  18720gcaagcttgc taaagaataa ctcatgttgt atttttggaa gaaaaaatat tt#aaataagc  18780agaaagaact tataaggtat gtgtacttga cttgcctcca aggacacttg ga#gagtgaaa  18840aattcctgcg tcgttgtgtt cagtgccagt catttaaaat gagcatctct gt#gctgagaa  18900acaggctttg ttctaagagc agccagttag aaagacacac tgtgtttgac ct#taacagtg  18960ggttctcaga aaacctggtt atattccttt tgcaccttat tcttaaaatt ct#gtacttcg  19020tgataccttc tgacagtcaa gtcaatgttc tgctttagga tgctatctaa gc#accactaa  19080attcactcac ttctctttct ccgctgtttt atttagcaca cagacctggc cc#agtatatg  19140tctcagcatc caggagggct tcatcctcat aatgtcagag tgagtacgtt aa#gggtcagg  19200accctctcct ggcttgccca cagaaggaga attctgaaac agactgtctc ac#aaagcaaa  19260gtcctatgat actaaataag aggatggaca tcactgatat tccagaaaaa ag#ttttgttt  19320tgttttcgtt tttgtttttt tttaaaaagg aaagaaaaaa gaaaaagagt tg#ctgagttg  19380cttcttaaga tatggagcaa tgttttctga gcaacctaat gctgtcagtc at#ggctacat  19440gcaaatgtgc ctttagatga ataaacgagt gaaggagaat tatactaaaa gg#aaaaaagt  19500aaagctaggc catcaaaaaa taaatacctt cttcatatca gattactgtg gt#ctaaggtg  19560aagtctgcaa tacttgtact agcagatcct attatatatg tggccctaac tc#ccattttt  19620ccagtcatta gaatcaaaat aataaactct taattagcta taattctaca tc#tgttataa  19680attttagaaa ccatttatat ttcatacttt tcattcccta aggttttatt gg#cattaatt  19740aattgattgg ctcttaaaat aaccgtatga aatttgtata tgatgtattt at#tcatttaa  19800ctaatattta tttatgtatt catttattca ttcatttaag aaatatttat tg#agtactta  19860ttgcgtaata agttctgggg tttcaataat gaataagttc tgtttcttat tt#tcaatgag  19920cttaaagtcc agtaagatat atgaacttaa ataggcagtg agggccagtc tt#caagcaac  19980agcaatgcaa gatggcagcc accatgggct caggcaattt atgaaagcca aa#tatacagc  20040cttaaaatag aatgtggacc taaataccca gaagaactcc cctttgtaag at#ttgtaaca  20100aaaattaata tgagtagagt taatagttct aatggaatgg tgaacccaag ag#ccatatca  20160gcgctagcaa aatggcagaa ttcatatatc atcaaagtta tccttcaaga gc#ttcagcgc  20220ctaatgatgt ctaaagaaaa tgtgaaacgc cctcagccat ctgaaggaca gt#gttacagc  20280aattgatcaa aaagaaaaac cacaggccct tccccttccc ccatacttga tg#taagcagt  20340cttcattttc catagtagta aattttctag atacagcttg tagagctcaa ag#tactggaa  20400agaaagctcc cattcaaagg aaatttatct taagatactg taaatgatac ta#atttttgt  20460acatttggaa tatataagtt gttagcctgg cgcggtggct cacgcctgta at#cccagccc  20520tttgggaggc cagagtgggc agatcatgag gtcaggagtt tgagaccagc ct#agccaaca  20580tggtgaaacc ccgtctctac taaagataca aaaaattagc caggtgtggt gg#cgcacacc  20640tgtaacccca gctgctcgag agagtgaggc aggagaattg cttgaaccca gg#aggcagag  20700gtgcagcgag caaagatcac accaatgcac tgtagcctgg atgacagggc aa#gactccaa  20760ctcaaaaaaa aaaaaaaaaa agaaatatgt aagttgtgct ataacaaata aa#taggcagt  20820gagaagcaaa gtgctaaagc ctatgaccat ggtaactagg aatactgtgg ga#acacataa  20880taagggaacc taacccagtc ctggaagtaa ggttttggaa aggaatgttt ga#ggacaaag  20940ggttaaagag agtgaaaaaa aaaattaaaa taccagttta gctgtgtgga ga#atgggata  21000gggagctaac tagagaaatc aaataggaat gtttcatggt atgttaagga cc#ctggtaag  21060ggtgaagacc attacattat ctgcaccatc gcgggacttt ttttttatgg ta#atgcttgg  21120caatttaaat agaggagcag agaatgtaga cagttggatt gagtcagagt tg#aagttctg  21180ccagacatgt gaaaggaaga gacaggtagg caagagagtt gaagagatta tc#aagacaga  21240agttaatgtg ctggccagtg gcatctagtc tgagtctaat ctgagggaag ga#agtgaaga  21300taagcagctt gctgatagtt atgaagagag tggaaggctt caaggaccta ca#ggtgttga  21360ttaaatagaa gaatgattgg agaaagaata actgtgagag agtgagattt tc#aggcttga  21420gtgactctca cataccagac actgtgctaa atgcttcaaa gacatgatcc ct#gccctcaa  21480gggacttaca gccaaaaaca agagataaga aatacacacc aatactatta ta#ggacactt  21540gtgtagaata tcaagaaaga aatacgatct agtactgtag atgtgcaacg gc#atcaaaga  21600tatcttctag tttcaagaag tttcagatcg gccgggcgcg gtggctcacg cc#tgtaatcc  21660cagcactttg ggaggccgag gcgggtggat cacaaggtca ggagatcaag ac#catcctgg  21720ttaacacggt gaaaccccgt ctctacaaaa aatataaaaa attagccagg cg#tggtggcg  21780ggcgcctgta gtcccagcta ctcaggaggc tgaggcagga gaatggcgtg aa#cccgggag  21840gtagagtttg cgtgagccga gatcgcgcca ctgcgctcca gcctgggcga ca#gagtgaga  21900ctgcgtctca aaaaaaaaaa aaaaaaaaaa aaagtttcag atcttaaaca ca#ctgcattt  21960caacagtcta gaataggaga gcatgttaca gggagagaaa atgttttcag ca#aaggtaca  22020gagtagggaa atagaggata tgttcaagga agaggacccc agagtcatgg tt#tgttaggg  22080ttagaggaaa cacagtgttt tgcaatctcc aggttccatt agtgcgttat ga#aatcaata  22140tggtggttag caacctgcat tttaaaaaat gaaataaatg gatgagaaga ga#atagaaaa  22200tattagcatg cattacattt tgaaagagca agtattattt tctgcaactt tt#gctccaat  22260tgtaactgta cttatatttt tatgtatgga tgtgaatacc agatacatat at#atttctta  22320ctgtagactg cagtcaaaaa atctttaaag cactggcctg gtctaacttc ct#tattttgc  22380agaggagaaa tccaagatct gagaggacaa acattttgcc tgaggttata ga#accagctt  22440atgccattgc taaaagtgat tcttagttaa aattctttcc cactagtgcc at#actgcact  22500tctagttctg ttggcctgaa atacagaata tattagtgaa acagcataca ca#agtctggg  22560gaaatatatt gggtaggtgg ctgagagcct cattttctaa gaaatgtgga cc#ttaggcag  22620ggtatggtgg ctcacaccta taattccagc actttgggag gccaagtcaa ga#agatcgct  22680tgaacccaag agttcaagac tagcatgggc aacatagcaa gacctcatct ct#acaaaaaa  22740tttaaaaatc agctgagcat ggtggcatac gcctgtagtc ccacctacct gg#gaagctag  22800gtgggtggat cgcttgacac aggagtttga ggctaaggtg agccatgatc ac#acaactgc  22860actccagctt gagtgacaga ggaagaccct gtccctaaaa aagaaagaaa tg#tggatttt  22920attccttaga cagtacagtc attagtcatt aagtttgagt tgagagaaaa ta#atatgatc  22980agaagaaatt tatatcactg tggtctgtag gatatatgaa aggaaataag ag#actagagt  23040cagggattcc acttaagtgt ttgtttgttt gttttgagac agagtctctt tt#tgttaccc  23100aggctagagt gcaatggtgc agtcatggct caccgcagcc tcaaactccc ag#cctcaaat  23160tatcttccca gctcggcctc ccaaagtgct ggaattacag gtgtgagcca aa#gggtttat  23220tgatgtggtc tggcctagtg cctctcaaac ttcagtgagc agacaagtga cc#gggaacct  23280gactcaacaa gtctgggttt aagcctgagc ctctgcattc taacatgagt ca#agctgatg  23340cagatggtgc tggtcaagag ccaagcactg agcagcaagg atctagttag ca#attagtaa  23400tcaaggttga tattatggta gtgacaataa gaatggagag gaatgtgaaa at#cagtaaca  23460aagaagagtt cacctcttgg taatgtgagc atgaggaggg aaaggatggg gc#caaacata  23520actggttttg tgtttgactg acgaggagaa ttgtagctct attaacagaa at#aggagaag  23580aagttggttt ggagagaaag aggagtcctg tttcagacgt gttgaggtcc ca#ggtgagac  23640aggatctcca aagggaaatg agcagtaggc aacctaaaag gaaatctgtg ct#cagaaggg  23700agctgtgagc tcgacgtgta gatctgaggg tcatcagcac atagagttta ga#agacaagg  23760agtaggcaac caaaagagca aatacacaaa gagaggagga ctgatgatga ga#cttttgcc  23820ttttaggatg agaagaggaa caggaaatga aggaatgaag ggaagcagct tg#taggaatg  23880tagagcatct gaaaaaaaaa tacacactgt catggaagtc aagggaagaa ga#atttcaag  23940aaggagggta tggtggacag tattacaagc atcaggaata cagctaaaag tc#atactctt  24000gactgcattg accttgtgga tttgtgaggg acacactaat aaataaagga at#ttattgtg  24060ggtatatgga ggcacaaagg aagaggttat ccaaatcaaa gcaggtggga gt#agggatga  24120gttctccaag gtggaggcat cagtgaatgt gggaaggggc acagagcatc ca#tgcccatc  24180ccaggcaagc caccctccag aagcctccat gagagttcag ctatccagaa gg#tctctgta  24240ccctaatctt tctgggtttt gcataggctt cattgtgtag gcatgattta tt#aaactatt  24300ggccactggt gatcaactta accttcaacc cctctcccct ccctaatcat gc#cttggtct  24360ttccagtgac cagtccctat cctaagctac ccaatggtct gccagctatc ag#tcaactct  24420acaaaaagac atcactttgg agattctaag gattttagga gttggctgtc ag#gaatttag  24480ttgaagatca aatatatatt tcacaatatc acagtcgtgc tattttatat ca#ggcgccat  24540taaatggttt taaacaaaga ggtgataaat tcagattttc tttttataaa gc#ttacactg  24600atgacagtgt ggtgaataga ttgggatgag ggcaatactt tttttttgaa at#gttatatt  24660cccctgaccc tactttctcc ttgttttctt ctacctctct ccccctactc ac#acagaaaa  24720cttctctccc tctactcatt ccctgaatgc tggtgtctgt taaggttcca gc#cttgacag  24780tgaggctaat cagaaccaca gtggtacaga tgtgagatga tggtgggaga aa#gtggacag  24840atatgagacc aattacttag ccggaactga cgggaaaaac aagagtcagc ga#tatttttt  24900tctggatctg agtattaaaa tggatgatgg tgccattcac tgtgatagag aa#tcagaaag  24960aaaaatttat tttggagaga taccatgaat tgtgttttag acatgctaag tt#tgaggtga  25020ttatgggatg tacaggcgag ctccagactg tgtgggccta aagtagaaag gc#aatctgag  25080ttggagataa agattttgaa atcatcagaa tacggttgtt cattagagca ct#gtcagtgg  25140gtaagatagc taagggagca tgtgtagagt gataacagaa gatcaaagac gg#aaccctaa  25200gaataacaat atgttattat ttattatttt attatgtttt attttttaat tt#tattttta  25260tttatttatt tatttttaga cgggagtctc gctctgctgc ccaggctgga gt#gcagtggc  25320gcaaactcag ctcactgcaa cctccgcttc ctgggttcaa gggagcctcc tg#cctcagcc  25380tctcaagtag ctgggactac aggcacccac cacctcacct gactaatttt tg#tattttta  25440gtagagacgg ggtttcacca tgttggccag gctggtcttg aacttctgac ct#tgagtgat  25500tcacctgcct tggccttcca aagtgctggg attacaggta tgagccactg tg#cctggcct  25560atttttgttt tttatagaga tggggtcttg ctatgttgcc caggctggtc tc#gaactcct  25620ggactcaagc aatcctcctg ccttggcctc tcaaagttct gggattacac at#gtgagtcc  25680ctgcgcctgg ccagaatatc aatatattag attttagtag aagtagaacc ta#tgaaaaga  25740acagccagag gggcagaaga aaaattagga gattgtggaa ccaaaagaag ag#agtgcctc  25800aggaaggaag gcatggtcta tgatgccaaa tgctgcaaag ataaggaata ag#aagtatcc  25860attgggtttc ataggaaaag tcatgggaaa ccatggtaaa aaaacattgt ga#atgacaca  25920atcgttgcaa aagcattttt atagggggat gaattttgta tttcagagga ca#aacagttc  25980catacaatgg caagatctag tgtgtgacca cgggagttag tgtctgaagt gg#attggaga  26040agcagatcat tggagctgag gttggctaga gctgttctca tggacactaa tg#tcatggag  26100tcaacagctg tgatccaagt gcccacatct tcagtgaatg acagagaggg at#tgagagtt  26160cagtgaatga ccgctaaaag aagagtaatg gaagatgtgg ctggatggca tt#aaaatcca  26220agggacaggg gtttttactt aaaagtagag aagtaatggt tttgaagtgg ta#gtggggaa  26280aagggaggca gcttatgaca cttgtcagtg gtcaaaggta tgaggaagtt at#agaaaaac  26340taacatccac ttgagaatat tatagggaag cagtgagctc aaggtctcat tt#aaggaaag  26400gagccaaaag gaaattcacc agaggttagc ttttaggtag tttttaaagc ag#gattgaag  26460aatggagact aaacagtgaa aatgtttggg agagagagga gcaatagata tg#aggctaaa  26520cagaggaagc acagaacaga atggagatga gtatgttggg aggaaaagga at#agtcagag  26580gcttatattt tgagttgtga ccaaggaaga cagggtggga atcctcgtga gg#ttatcttg  26640tttcagattt ctagtagaat gagtcccagg gattccaggg gggatggaag ga#ctcaggct  26700tccctataag gagttggcta acggatctca ttggtttttg agtaactcct gg#cccagatg  26760gcactagttc aatggaatta ttttgttccc ccaaaactta ttgagttgga aa#caggtcta  26820actcctggga tctgggaagc ctttctggaa agagtcaccc acgatctggc tg#atgttgaa  26880ctgtgcagac accatcatat ttggttatgt taggatgcaa taattggtga ag#cttctgta  26940gtgttgaatg aagaatccag gttggaaggg atgaaagggt gagtgggtga tg#aggtttgt  27000cagcacagac tgcaattttg agaaatgtgg ttataaaata ccatacctta at#accgcagt  27060gctttaccac tcacaaatgc ctgtagacgt atctggcaga gaggaaaggg gt#tgaatggc  27120aagaatgtgg gaagggactg tggctagtta gtgaaaatag tctacacttg gg#acataaaa  27180ggcatttcaa gctgacctac taagaagctc tgtctctgac tcagccagct gg#ctctctcc  27240ttccctgtca tgttttcatt ttctgtcttt tctctagttt ctcaggatgg ta#tagtggag  27300tcagacaagt ctgaatttga gtcttggctc tgactattcc tagacatgtt tt#aaaagtta  27360cattgagccc tggttttctc tgtaaactga ggataagcat gctatcccaa ag#gttgtatc  27420cctcactggt caccagcttc ctgtcttcta tccacctgtc ttcctcttcc tc#tttcccta  27480gtcctgcata ttgaaaaaca tttttttttt tttttgagat ggagtcttgc tc#tgccaccc  27540aggctggagt gcagaggcac gatcctggct cactgcaacc tctgccttcc ag#gttcaagc  27600aattctcctg cctcagcctc ccgagtagct gggattataa gcatatacca cc#acatctgg  27660ctaatttttg tatttttagt agagatggag tttcaccaca ttggccaggc tg#gtctcgaa  27720ctcctgacct caggtgatcg gctcgctttg gccttccaaa gtgctgggat ta#taggcgtg  27780ggccactgcg ccagtctgaa aaacgtattt ttaagcacat actatcgtat ct#tcttgtct  27840tttacctgga atttaagctg gttgtttgta ttaccttttc catggacatt ta#tatttata  27900accaatcaga aggtttaaat gtcagtgtag gaattttgtg ctatggaagc tt#cgtggctt  27960ggtgaatggt aaaatgaata atgtgtgtat atttgaagca tcagaaagag aa#aatgctgg  28020gaagattcat agaaccagtt aacatttgaa ctaggagtca taagaaattt tt#aaaattct  28080taaatggttt atgaacctga tgtggtagct acatgaaacc tgcatagctg ca#ggtatgct  28140atggtaggta aactctccat gctcctgctt ccattggacc atttggctcc aa#tgtctcca  28200ggtctttgtt agatcaatac tggtcctagc atctctgaaa gtcctagctt tc#taagatgc  28260tgttgaaaaa gaggattaat ccacataact ctgcatctgc cattttgccc at#gtcccagg  28320aatgctgggc ctagcccttc ctttctgaac tgccagaaca cgttctcagt tg#acatacgt  28380ctttgtaaat actgatgttg gtgtttgaat tctcaattgc caatggcact gg#aaaatagc  28440aaaagatact tggaatacta agcattcttt ttttcccgta agtttctgta gt#gatgggaa  28500cctagtaatg gctttggttt ctgtgcctca taaccacatg aaacattttt aa#tttggggc  28560tcagaatgtg tttttccctt ttatttctcc accactacca tttacccttt ct#cccttctt  28620cctcctacaa tttgttcctt attctttttt gatttttttt gagggggggg gg#tctaactt  28680attttggtct ctcttccctt ttcatctgta ctgtgtattt cccttgtttt ca#actttgaa  28740tttaagactt taaaaatagc tttaaaaaga taaagatttc tttattttct aa#taccatct  28800aaagatatat tttttagtgt ggtctccttg tgttgtgttt ttaaaagggt tt#catattgg  28860agagcctgga aaacttaagc agttgtaaac tttagaatat catttccagg tc#aactttga  28920tcttatatgc caagttcatc ggtggggaaa aaaattaaat ctttcacatc ta#aatcaata  28980actagtgttc caaaggaaac ttcaaagttt cactttagat ttttaaagaa gg#gtaattcc  29040ttcagtatca aagaaatgag atgtcaggaa aagccagaat ccctttgttt ag#gacacagt  29100ctagttactt gacttttctt gtcctttttc ttccccctct gaatgtaaaa at#cttcttct  29160tcttcttttt tttttttttt ttggtctctc aagagacact tttactatat tc#tttgagat  29220gactgttttt gatttagagg cgaaatcagc acgtggtggc tcaaatctcc tt#atggatag  29280tgtttcttcc ttccagcttt tcatgtttca acttttgcgg ggcctggcgt ac#atccacca  29340ccaacacgtt cttcacaggg acctgaaacc tcagaactta ctcatcagtc ac#ctgggaga  29400gctcaaactg gctgattttg gtaagtcgcc cctcgggtct cattctgggc tg#tgaacaat  29460gatgcttttg tgtgcacttg tttaagcgtt gactgggcct ggcctttgaa aa#ctggaggc  29520ccaagaacat gatgctttgt gaggatatca aactaccaca aaggaagtgt ga#ggcacgaa  29580acagggaggg attggtagct ttctaggatt ccaccaagtc ccagtttagt ca#gatggcca  29640aaagctgggc acccttgctg ccccactgcc agttttgata tagagacatt gg#tagagtaa  29700actgtactta gtaagttttc ctaaatctaa gtgaatatac aaattatatt gg#aatagatt  29760gagattatcc caagatgata aagaggttaa ccccagattg tagcatggac tc#ctgtcagg  29820atggagactc caggacactt gttcctgctc tcctaccttc tttatataag tg#tgagatgc  29880aaagttttat tcccattaaa gtgaagcaga tttcctctaa gtatcactgt at#ccttccat  29940tttagcactt atcgcagttt ataattatat tcacacacat aaatacatac at#gcatacat  30000acaaatatat atacatgtgt gagcacaccc ccacacacaa atatatatag at#ttgcgtga  30060tgattttgtc tcaactggac tgtaagcata atgagggcag cctgggtttg tt#tttgctta  30120tcattttatc cttagtgcct ggtaccatag taggtgctta ataagtactt gt#tgaaaaac  30180tggctctatg tgagctaagg aaccactctt ctctgtttgg cagatgccaa at#ggtgatac  30240tatcactgca gtatttattc tgagatggca gcttttatcc tgacatgtaa gc#atttaaca  30300gatatttgtt tatcaattct ccacaatagc aaactcatct attgaagttt tt#cccaacaa  30360tagatcatgc aattctgtga gataaacagc tgactgacag aaagactcat tt#tgcagaac  30420agtacttaga aattcatcta aggtcctacc aaactaatta atttggatga gc#agtcccta  30480ccgtttatct actaaactgg gctttcctgg agtgccaaaa cggaaggtgg cc#atgttagt  30540catgaacagc tcagtttctg ttacagagac ccaaaattac agaggtataa ca#tgctagaa  30600acttaacttt ctttcgcatc acagtcctga cctaagcagg cagagcatgt at#ggtggccc  30660catgctatct tggcccaggc tgcttctgtc acgtggctcc tccatcccca at#tgtatgtt  30720tcaagatggc tgccacttcc tgctcatcac agcccagagg agggagaaaa ga#gaagcaga  30780acccttaacc cctccactaa ggcataatct ggaagttcac acatcacctc tg#ttcatatc  30840atataggcaa gaacttagtc acctgaccac acccagctgc caagaaggcc ac#atctagct  30900gcaaagcagg ccaaaatttg agaaattcac ttgatgaagt gatagacaag ag#tcaagata  30960gtgattagtt ctactaaaag cacctaaagt ttgtgtgtta ttttttctaa tg#gtgtttac  31020cctggtccag tgcatcatgg tgcaagccaa ggtccagaac gatgggtttt at#gcttttcc  31080cttttggaca ggtcttgccc gggccaagtc cattcccagc cagacatact ct#tcagaagt  31140cgtgaccctc tggtaccggc cccctgatgc tttgctggga gccactgaat at#tcctctga  31200gctggacata tggtaagagt ggtgccgaga aaatgtgagt catcctactc ac#gagggttg  31260ctttatcatc tacattatat tttaataata attctaaaaa tggcaatcac gt#atatattt  31320ttatatatat ttatatttat atattttata tatatttata tagttatata tt#tatatttt  31380atatatttat atatttatat atatttgtat atatttatat atttatatat tt#ttatatat  31440ttattatatt tatattttta tatttttata tatttatata tattttatat at#atttatat  31500atatattata tatatttata tttatatata tttatatatt tatatatatt ta#tatattta  31560tatatattat atattttata tatttatata ttatatatat tttatatatt ta#tatattta  31620tatattatat atattttttt atatatatat atatgtattt tttttttttg ag#atggagtc  31680tcactctatt gcccaggctg gagtgcagtg gcacgatctc agctcactgc aa#cctccacc  31740tcccagattc aagcaattct cctgcctcag ccttctgagt agctctacta aa#aaaatact  31800aatatttgta gaagattctt gcaattattc tataaccttt tactgttgaa ct#gagaccca  31860cagagttcct gcccaaggca tcttctgaat ctgacactct ttttatgtta tt#ttattttt  31920tgagattggg gtcttgctat attgtccagg ctggtcttga gctcccaggc tg#aagcagtt  31980ctcccacttc agcctcttga gtagctggga ctatagggct gcaccactgc ac#cctggcaa  32040tctcatgctc tttctttcac gcctttcctc ctagctcctc tctttaatcc tt#tgccttgt  32100cttctccttg acaccttatc cacagagaaa caaacatata tccccaaacc ac#agacacac  32160agatgtgtgt gcacgtgcat gtgcatgcac acacatctgc atgaacatac tc#acacatgt  32220ccaaacgtag ttcagagcct ggtttaggaa aaaaaaaaaa aagcataaag ac#caagcttc  32280aagacacctg attttcatgc cagttcgatt tctaatcaat taactctgga tt#ctgttatc  32340ttgaaaaagt catgtatcct ctctgtgtct atgtttctcc atttttaaaa at#gaaggtaa  32400taaactctct ccatctgagt taaatggaat tgtagtacaa atataagaac ca#aataggtg  32460gctgggcttg ccgtctcatg cctgtaatca cagcgctttg ggagaccaag gc#tggaggat  32520cgattgcttc agcccagttg tttaagatca gcctgggtag cacagtgaga tg#ctgtctct  32580acatttttta aaaaaattag tcaggcgtga tggctaatta aacacttcag ga#ggctgaag  32640taggaggatc tcctgagcct gagaaattga ggctgcagtg agttttgatg gt#acccctgc  32700aatccagcct gggttacaga gcgagacccc gtctgaaaga aagaaagaaa ca#gagagaga  32760gagagagaga gagagagaaa gaaaggaaaa gagaaggaga ggggagaggg gg#agaaaggg  32820agagggggag agagggggag aaggggagag gggggagagg tggggaggga gg#gagggagg  32880gaggaaggga aggaaggaag gaaaggaagg aaggaaggaa ggaaggaagg aa#ggaaggaa  32940ggaaggaagg aaggaaggaa ggaaggaaag aaggaaagaa tccagatagg tg#ctatcaag  33000taaagccaca gagttgggga ggctctaagg ttaatgggtt acaatagtga gc#atgggctg  33060tcagacatgc atcatcctag aacggcagtg ttattttctc tggatcatgt tc#ctggagac  33120ttcccagtca tttgggggcc actgttagat atgtgatgac tttacagacg ta#gacaactc  33180cccaaaggta aggaaatata tgaatctctt tcagtacctt ggaagaaagg gt#ttatataa  33240aaacacaaag ccccattttc aaaaatccat aattgatttt aaaaaattaa at#ggtgtcct  33300aaaaggctaa actaagcttt tagatctccc aaagaattaa gaaaggttgc ag#acattttt  33360ctccagtgta gagtcattga tttctgatac ccagtacaat ttatagaaat at#catctgct  33420agtcaaaacc ctcctgaaac tgtcagctca caccgctcag cactgtcact tc#aaaggact  33480ccggcaggct ctggcttact cagctcttaa tgatgtcttc ctgattatgt tt#cacagagt  33540gaaacttcta cccgtcaatt ttaaactaat tttattatgg aatagttaaa ac#attcaaga  33600gtatatataa catatatgta gatcagtgat tctcaaccag ggagcaattt tg#ctctgcag  33660gggacatttg gcaatgtctg gaaacatttt ttgttttcac agctgggggt gg#ggtggtgg  33720ggggtatcac tggcatctag tgggtagaga ccagggatac tgctaaacat cc#tacagtgc  33780agaggacagc ccctgcaaca aagatttttc caacccaaaa catctgtagt at#caagatta  33840agaaagccga tgtaggttaa gaagcttaat ttacttttag agacagggtc tc#ccttggtt  33900gcccaggctg gagtacagag gtgagattgt ctcactgcag cctccaactc ct#gggtttaa  33960gtgatcctcc tgcctcagcc tcctgagtag ctgggaatac aggtgtgtgc ca#ccacacct  34020ggctaattaa aaaaaaaaaa gtgtagagac agagtctcac tttgttgccc at#gctggtct  34080caaactcctg gcttcaagag atcctcctgc cttggccttc ccaactgctg gg#attacagg  34140tataagccac cgtgcccaac caattaagaa gcttaataac gtgaacttca ta#acctgcta  34200cccagtgtaa caactagaac ataatccgta ctgtcctatc aactgtgtcc ct#ttcccatc  34260aacctgcccc tccactagaa ggccttctac caaaattttt tttccttttt tc#atcagtat  34320tctcatatct ttttaaaaat aatcctttta cattttagag gtattcttaa aa#atattttt  34380ttgttttact tgattttaag ggttgttttt ttttgagacg gagtctcgct cg#tcgcccag  34440gctggagtgc agtggtgcga tctcagctca ctgcaagctc cgcctcccag gt#tcacgcca  34500ttctcctgcc tcagccatga tgttatattg cttctagtct tctgtgactt gg#ctttgttt  34560cattcaatat gttacatgtt tctaagattc atccatgttg atctgtttag ct#atacttta  34620ttttctgtta gtgaatattt catttttttt aatgtctata gctttgcaat aa#tacttgat  34680accttgtagg ccaagtctcc cagcctattc atcttcttca tgaggataca tc#agataaac  34740ctagtttaag ggacattcta cagagtaact gacctgtact tattggaagt gt#caagattt  34800taaaagataa agactgagga actgttccag attaaaggag actccagaaa cc#tgccaact  34860aaatgtaacg catggtccta gattggatct tgggggagat ggtgctctaa ag#aatactgt  34920agggactata ggtgaaattt cagtagggac tgtggattag ataggggtat tg#gatgaatg  34980ttaaatttcc tgattttgat aattgcactg ttgttatgta agaggatact tt#ggttctca  35040gaaaatacca acataattat ttagggatga agagtcatga tatctacaat tt#actcccta  35100atgtttcaga aaagatatag acagacagac agacagacag acagacagat ag#atagataa  35160aataacgaaa caaaagtgac aaaatattgg cgatggatga acctgtttgg ag#gatataag  35220agagttcttt atactgctgc aacttttcta taagtttgaa attatttcaa ga#ttaaaagt  35280tgcctccaaa ttgcgaaatc cttgctgttt catcaaagtt agtgtaagac ag#cactagcc  35340taatatgtga tcagtgtttg taatttcttc atgtgtgttt gagaagaatg tg#tgtgtcca  35400cccaaatgtt gagtgctgct ggggtttttt ttttgttttt gtttttgttt tt#gttttttt  35460tgagacagag tctcactctg tctccatgcc tggaatgcag tgactcaacc tc#ggctcact  35520gcaacctcca cctcctgggt tcaagcgatt ctcctgcctt aacctcccaa gt#agctggga  35580ttacaggagc acaccatcac acccggctaa tttttgtagt tttagtagag ac#ggagtttc  35640gccatgttgg ccaggctggt ttcgaacttt agatgtcagg tgatcagcct cc#caaagtgt  35700tgggattaca ggcatgagcc accgcgcctg gccaagtacc catttttaca ta#tgttcaaa  35760aattcaaggt tgctaattat attatccaaa tcttctttat attatttttg tc#tttttaac  35820ctaccaatga aaggtgtgtt gaactcattc actatattgt tgatttgtca ga#attctatc  35880cacttttgct ttatatgctt tgaagctatt ttcactaagg gcaaataaat tt#aagactgc  35940tcattattcc tttgtacact ttagttacca ctttcagaat aattttcatt tc#tcctgaaa  36000tacatctttt agagtgtttt gttttgtttg tgtgtgtgta ggcctgctgg tg#gcaaattc  36060ttcgtttttg ttttcagaag ataaacccta attattgaaa ggtggttttg tt#ggggatgt  36120gattctagac tgacagttat tttctctcag aactttgaag atgtcattcc cc#ttctttgt  36180cttccattgt tgctgtcgag gagtttgctt ttagccttat tatcttcctt tt#gcaggtga  36240tctcattttc tctggatgtt ttaaagactt ttttctttgc ctttatgatt at#gcagtttt  36300ctctaggagt tgtccagtgt ggatttcttt ttacttaccc tgtttggtat at#cttgtgtt  36360tcttccattt gtgaattcat gtctttcatc agccattttc tttttgaata tt#gactctat  36420tctattctct ctctgtagag ctccaatgaa agactattag accacattct tc#tgttatcc  36480atttctcttc tctccttcat attttccatt tccttaactt tctgtgatgc at#tctgggta  36540atttcttcag ctcatctacc agttctttaa gtctctctta aactatgtat ta#ggttggtg  36600caaaagtaat tgcagttttt gccattaaaa gtaatggcaa aaccatagtt gc#ttttgcat  36660caacctatat ctcttacctt tttaccacat atacaaaaat gtatgttatt ct#atgaataa  36720gtgtttcatg aatttaacca tgagcaacaa tgacacaata taaaaatgca gt#tataagtc  36780aaaattattg ttattactct tattcattcc atttgattgt tgttttcctg gt#aaaactaa  36840aaatgtaatg tagaaataga acaatatgca tcttccattg agctcactat at#ttgtttac  36900cctcaaagta attgctagac cttgggtatt tacactgaga tccctctcct cc#catttttt  36960tctttttctt ttcagagtga taagagggga agtgagaagg gagaagattt cc#agttgaca  37020aagaatgaaa aagaaagaat aatcctattc tgctaggcca tgcaacccca ta#gggtccaa  37080agtgaatgcc cttgtaggag gtagatgaca ctgggtgagc attagtgcat tt#gtcttaaa  37140gaaaccaatt ataacccgta gtgcagagcc tctccttcac aatgaggcct gg#tggcagca  37200gtgtcagtag ggggccagag caaataaaca ggggctctag ttaattatgg aa#aacttgca  37260actaggacat attggttatt cccaaagctc ccaaccaaca ttctctcatc tt#ctgacgtc  37320ttttcttctc tctctttctg ctaccttttc agaccttaaa agattccatt ag#tgacttta  37380gtgagaaaaa tgcaatattt taggattatt aaatggtgtg gtttttagtt tt#ttgtattg  37440tgttaaaata tacataaaat ttaccattca tcacgatttt caggtgtaca at#tcagtggc  37500attcagtaca ttcacattgt tgtgtaaccg tcaccactgt ccatctccag aa#cttttcat  37560catcccaaac tcaaactctg cacgtattaa atgataattt cccattaccc cc#tctcctca  37620gtccctggta accacgattc tgctttttat cttgatgaat ttgactattc tt#ggtacctc  37680atataaaagt ggaatcctac aatacctctt ctgtgtctag cttgttttgc tt#ggcataac  37740attttcaagg ttcatccatg tcgtagtaca ctgagttttc cagaagcatt ta#tttcagta  37800cacaaggtca tctattcagt atcagtttca ggcagctgct ggtgttagga ct#agagaaag  37860ttgtctctgc ctaacagatc atttactgtc acatttctcg ctgcaaactt cc#aaatataa  37920aaagggtggt ctagagaaaa gcaagtgaga atgtcatgtc actgccatat at#tacgttat  37980tctgaattaa cttcaacagt aagaaatgaa atactgattc atttctccca ac#aacatttt  38040gatattctcc ttgcacctcc aaaaagccta aaactcccga gatggatttt tt#ttctccag  38100ggactgccta aggaatctga ggaatctttc cccctcttat ggaagaattt gt#tcatgctc  38160agaatagaga aaaagtagga ggagaaccag aaagaggaga aaacatctaa gc#agtttcct  38220ctaacttgac tgaagaacca catttggaac aataaaatga cccagcacat ct#ctcccttc  38280tggaagggtt taatgtttga tgtcacaggg tcttttctcc cctgcatatg aa#tttcccct  38340tcgtctacac gggctgcccc acgggtatct ccacacagca gaaatcctca ga#gaagctta  38400aagatatgta gggtaagagg agccccagga atgaagattt aaggacaaaa ca#gaaaaata  38460aaaggaaata gaagctggtt ccctatctgg acttgaatgt tcagaatatt ta#aaatgttt  38520gctttaagaa tagtctgtgg tgggcaaaat agatgatagc cacatgactt gt#attcctaa  38580gggtaagaag caaattaaaa aaaagaaaca gttctgaaca gaaatgaaaa aa#taagataa  38640attgcatagt tctttttttt tattagatgg agtctggctc tgtcgcccag gc#tggagtgc  38700agtggtgcga tgtcggctca ctgcaacctc caactccccg gttcaagtga tt#ctcctgcc  38760tcaacctcct gagtagctgg gattacagga acacaccacc atacccggct aa#tttttgga  38820tttttggtag agacggggtt tcaccatgtt ggccaggctg gtctcgaact ga#cctcatga  38880tctgcccgcc tcggcctccc aaagtgctag gattacaatg cttacaccta ga#acagatct  38940gtcacctttc aaacttacag tgtgggcttg ttttgttatc aatgcattga ta#tttacagt  39000acctatggat agtccatgta ctgaaataaa attgatttag gaattttgtc tt#ataagtgt  39060tctaaagact tgcacaagtg cacacataca cacactatat acatagtgtg tg#tgcatgtg  39120cgtgtatata aatgagtaac cttagactta gatttgttag atgaggaagg tt#tcaacctt  39180ccccaaaatg caaatggaga atttcaacca tataaaccaa atattggcat tt#tatctctg  39240gaacacaaac atcttgtgtt actttatggt acttacgtaa tggcctgaat gc#tctagttt  39300ttgccaatat attttacata attttgtata caagtttagt ggtatagaag at#aaaggaca  39360ctaagcagga ttaacagctt ggttccctac agctgttaag tatgaaaaca ca#ccatgaaa  39420aggcaacaag cttcttccag gcaatggaag gctttttggg ggagaaaaga aa#gtgaatta  39480caggtttaaa cctaggaatg tcattttttg aaacttgttt aaaatatttt ca#atccttct  39540agtggtttgt gagctcctgg ggtttctgga aggtgtttgg gaactggata ga#gggttagt  39600tcatgccttt aaaagccaat acatttccat ttctctttta taaccaagta at#aacccaat  39660tatgcatgta ttttatatac acagacacgt atttattttt actccaaaac aa#aatggtct  39720gaggcctttc aagaaagtgc atgtggcgaa gtcatggggg gcagggtgga ga#ccatttgg  39780tggtgcccac taactaggtt tctcagttgg cttatctctt agtggaccat tg#ctagcaac  39840cagggtgttt ttaagcattt gacagttttc catcactttt atttgccttc at#atattgtt  39900tcatttacac ccttagtatc tcttgtttta aagacaggag acaaaaagaa ca#tggatatt  39960taaatacaag ttaatgagga actttaaaat aataataatt ctacaaattt ac#ctcaagat  40020actttaccaa attcataagt tacatttatc tgatcaaaat tcttgtgtca ca#tatcaaga  40080tgtttcttat acagcagaaa tcagtagaaa agaaaaaata ggccaagcgt gt#ggtggctc  40140acacctgtaa tcccagtact ttgggaggcc aaggcaggag gattgcttga gg#tttggagt  40200tcaagaccag cctgggcaac acagtgagat cccatctcta ttaaaaaaat ta#gaaaagaa  40260aaagaataaa atggggctgt tatatccaaa ttggcttttt aaaaatcagc aa#taaggccg  40320ggtgtggtgg ctcacacctg taattccagc actttggaag gctgaggcag gc#ggatcaat  40380tgaggccaag agtttgagac cagcctggcg aacatggtga aaccctgtct gt#actaaaaa  40440tacaaaaatt agccaggcat gctggtgcat gcctgtaatc ccagttactc ag#gaggctga  40500ggcaggagaa tcacttgaac ctgggaggtg gaggttgcag tgagctgaga tt#gcaccact  40560gcactccagc ctgagtgaca gagtgagacc ctgtctcaaa aaaaaagaaa aa#aaaaattg  40620gcaataaaaa caacctgttg cttgctggag gaaaaacctg cttgcaaagc tc#agtctgat  40680atcatttttt aaacaaaact ctaagaacaa gccagtcagt taagctaaaa cc#aaatattt  40740gattatgaaa agggtttttg tatattttta caggataaga tacaaataaa tt#tcagtctt  40800tcttttaata tgtatttctg ttcccaaacc agacacaaag caatttttaa ac#ttgatcgt  40860caagaaatct gttttctcct acacaatcaa tgaaaagtaa tctaaacagt gt#ttgtcagg  40920ccaggcacag tggctcacat ctgtagtcct agcattttgg gaggcctagg ca#ggtagatt  40980gcttgagccc agaatttcaa gaccagcctg gacaacatgg cgaaacccca tc#tgtattaa  41040aaaaaaaaaa aaaaaaagac catatgtctg cagtcagatg gaaaaagtaa aa#atatgtaa  41100taaacacata tgaataatat taaggaccat attttaaaat aaacttgata at#aaattttt  41160aataatatta tctacgataa aatgttttac ttaaatttcg ttctttatca tg#ccacacaa  41220aaatggcaaa atgattaaga gagtttgcaa aattatgtgg tatagtgaaa ga#ggtttgcg  41280gttaaaaaaa aaaaagagag agagagagag aagtatgggg ccatggggat ag#tctctgta  41340atcagtcacc tgaaccactt ttaatactca aaagacttat gagaataaaa at#ctgatttt  41400tgctaagatt tattagcaaa ataaatctta ctccttcctg tccctctcta at#tatccttc  41460agcttgacca tgtatgaaag aaaatttaca tttcactgtt taatctattt aa#agatgaac  41520atttcccatt aaatcaggat gcaccttata atcagtagca tctaacaata ta#agtcagcc  41580aggctgcagt tgtgactgta gttagaattg cacatgtgtg aacatcaaat ga#gccagcat  41640caaaacgtgc agaatggcca ggcacagtgg ctcacacctg tgatcccagc ac#tttgggaa  41700gctgaggtgg gtggatcact tgaggtcagg aattcaagac cagcctggcc aa#gatggtga  41760aatcacgttt ctactaaaaa tacaaaaatt agccaggcat ggtggcaggt gc#ctgtaatc  41820ccagctactt ggtaggctaa gtcaggagaa tcgcttgaac ctgggaggcg ga#ggttgcag  41880tgagctgaga tcgcaccact gcactccagc ctgggcgaca gaccaagatt cc#accaaaaa  41940aaaaaaaaaa attgcagaat tggtgtcagc gacttggaag aaaattctgc aa#agaaaagt  42000cctttttttt tctttttttt tttaaactcc taggaaccaa atggttgtgg ag#aaggagta  42060aatcagacat gtttagcaac attctttaag caggagtcaa aagtaagcta ac#actacata  42120actgcaaggc cagcttagga gcccaggacc aatgactctc tgttgtttta tg#gattattt  42180taagaaatgc tgcatcatca aattcttaat atagaggatg atacatgggt aa#gtgtagac  42240atcaaagagt ctgagtcaaa tgctgaatgt gaaaaagttt taggaatacc ga#aaccaatt  42300tattttgctt aatgtttctc tttttcgtgt acaagtatgc tatatgagaa aa#taatctct  42360atttaattaa atttataaca gccctttcaa taagtataaa atgaacattc tg#atcatgtc  42420atagtttaac ttgcattttt ttgtcttaat ggcaaaaaac caatgacgct tc#ttacaatg  42480atagcatctt agactcaatg aaaagtgggg atgaaatgaa atttggggat ac#agtacttt  42540cccctcttct cctaaaacag ataatgagct tgaatgatct acaatgtttg ct#aactctac  42600tgctttccta actgctgctc gtggtgttcc attttaataa aaagctgtgg gc#tgttctta  42660ttttgtttga catagggact ttttttttgg cccaagactt ttaatatcat gt#ggtccgta  42720tttaactctc cctaaaatat ttcttgggaa gagaaattct agtagttcag tt#tcgcttgt  42780atgatttctt tcaaagtgtc aatttactct tatttccttt gctaggggtg ca#ggctgcat  42840ctttattgaa atgttccagg gtcaaccttt gtttcctggg gtttccaaca tc#cttgaaca  42900gctggagaaa atctgggagg taggagaata attcttctaa agaaaatgaa at#atctgcat  42960tttaagtttt gaaccaaatt tgccttacag acaaatgaag cagtccatct gc#tctgagat  43020attaagccct atattaagat tgtagaaact gtagcatttg ccacagctat aa#gcaccctg  43080ggaatgtgtg gtcaggaaac tccctgttgc cccatagcag cccatgaatc ca#gctcactg  43140aatgatgttc aggtctcctg ctccctgtca ttagtattgt cttaacctcc ca#gggcaatt  43200tctgccatta ctactcagac atgtccctac cttgctacct ccagttctaa tg#ctaccata  43260tatttggccc tggatctttg tcaactgaaa ataagacata gaatttttag ct#gggtgcag  43320tggctcatgc ctgtaatccc agcactttgg gattgctttg agcccaggag tt#cgagacca  43380gcctgggcaa catggcgaaa ccccatccct acaaaaacaa aaatgagtgg gc#tgtgtggc  43440gcacacctta gtcccagcta ttcaggaggc tgagatggga ggatcacttg ag#cccaggga  43500agtcgaggct gctgttagct gtgaccacgc cactgcactc caggctgggg aa#caaaaaaa  43560agacacaaaa ttttcataga accctgatag aacagaggct ttccctctta gt#gtgaaaga  43620agtgtaccat ttatcatgct tatccacagc caaattccta aagtgtcaag gt#gcctttgt  43680gtgtgtatgc agctccattt cttaattcat tatttatccc taccgcagtt gc#ctatgata  43740tgctttgttt ttatggccct tatatagtat tacagtcata ctatagtcat ct#gtatattt  43800ccttttttgg tcatattttt attgtggtaa aatatacaaa acaaaattta cc#gtcttaac  43860cctccttaag tgtacagctt gtcagcatta aatacattca tatagttgca cc#accatcac  43920cgccatccat ttccagaact tctctatcat ccctaaggga agctctggac cc#actgaaca  43980ataactgccc atcttccctc cccacactcc cctagcccct agtaacctct aa#tctacttt  44040ctgtctccat gaattggcct attctaggta cctcatataa gtggaatcat ac#aaatttgt  44100ctttccgtat ctggcttatg tcacttagca tattttcaag gttcatccat gt#tgtagaat  44160gtgtcaaggg gctttaaatc ggcggggtgc aggggggtac tttattactt gc#tatcctgg  44220atcctgctgc ttgtcttctg gctaaaataa aatgtacttt gtgaaattaa ga#cattttat  44280agagattaat tactgacatt aaattttctt ctagaaacat gggggctatt at#gaaggaac  44340atgggaaaaa ctgggaagca ttcacaactg aaaaaaaaaa atccaagcca aa#agactttt  44400tctaaaaact ttcttgcaag acagagcaat gctatcttca cattatgtta tt#gggtgcta  44460taacatcatc taagctggag acagcctact gtcatagctt tggagtccaa ag#acctgggt  44520ttgaattcta accattttct agctaaatga acatgggcaa gttatgtagt cc#ctctgaac  44580tttcgtttcc ttgtctgtaa aatggcaaca atgataataa ggactttcta at#tctttatt  44640gagaattcca taaaaacaaa tgcataacaa gctccatgca ccataaatgc tc#aatagatg  44700cttgctttct tcctgtccca tacaaattgt tgtacagatg tttcaataac ct#aactgcta  44760gcaagtatta cctgaaattt aacccgattg ttctcttctt tcacttagca gt#attatttc  44820ttgtccacaa tagaggaagc acaattgcag ttctgatgct gcaatgacct tt#tatacatt  44880tgaagagttt ttcctggtca tttaatcagg aaacaacact tactcaccat at#atgaggcg  44940agtaactcta caagactcta caaggtcttg taagaagcta taagccaagg gg#gaaaaaaa  45000aaagaagaat aagaaaaaca catgatctgt attttcaagt gttgttcagt ct#aggtaggg  45060cgatgggtga agtatacgta aatatatgtg aaacaaacat aaactatgta ta#tatgtaaa  45120aggatgtatg tatagatagt taatataaat tgtaatactg aaataagatg tg#ctattagg  45180atacttgaag agtagtttat ttgaaaagaa tataagtata tccttgtgtg cc#attagtat  45240ttgaagagtt gtatataaac tgattttttt tctttttcct tttttttgag aa#ggagtctt  45300gctctgtcac ccaggctgga gtgcagtggt gccatctcgg ctcactgcaa gc#tccacctc  45360cccagttcaa gcgattctcc tgcctcagcc tcctgactag ctggaattac ag#gtgcccgc  45420caccacacct ggctaacttt tgtattttta gtagagacgg ggtttcacca tg#ttggtcag  45480gctggtctca aactcctgac ctcgtgatcc acccgctttg gcctcccaaa gt#ggtgggat  45540tacaggcgtg agccaccgcg cccagcctca taaactgatt tttaaaatac aa#tatacagt  45600taggcatagt tgtgtgtgcc tatagtccct actgcttggg aggctgaggc ag#gaggatcc  45660tttgatccca ggagtttggg caacatagtg agacccccat ctctaataat aa#taaatata  45720aatttcaaat aacattttaa aatatgacat actatctttg aatgaccaca ca#atttaaaa  45780agcaatcatt ttacggttct ttagtgttca gttagcacag cacttagaaa tc#atagaata  45840aagtgagcaa gatgcttctc aaagcctgat cactctttag gactcacaat gg#gctaggta  45900ctatgctgga aagagaaaaa ataataattt tctaacctgc ttgagacata gt#ggtataaa  45960tgataacaca gctgctgaac gtgatgactt tctcactttg tccgcagagc aa#gaaactat  46020agatgcagta acaaaactgc attcaatgaa catgggactg tagataacaa ac#taacttca  46080tttctttggg tacatgccct gtattgggat tgctggatca tatggtagtt cc#atttttaa  46140tattttgagg aacctccata ccatcttcca taatggctgt gctatttgca tg#cccaccat  46200cagtgtgcaa atgctccctt tcctccacat tcttgccaac acctctttca tc#tttttgat  46260aatagttatg aggcaatatc tcaccatggt cctagacttc atttgtctga tg#actaatga  46320tattgagcat tttttcatat atctcttggc catttgtagg tcatcttttg ag#aaatgtgt  46380attgaggttc ttagtccatt cctgctacca taacaaaatc ccttagagtg gg#cattttat  46440aaagaacaga attggcccgg ggcgcagtgg ctcatgcctg taatcccagc ac#tttgggag  46500gccaaggtgg gtggatcacc tgaggtcagg agttcaagac cagcctggtc aa#tatggtga  46560aaccccatct ctactaaaaa tacaaaaact agccgaacgt ggtggtgtgc ac#ctgtagtc  46620ccagctactt gggaggctga gacaggagaa ttgcttgaac ccaggaggag ga#ggttgcag  46680tgagacgaga tcgtgccact gcactccagc ctgagcaaca gagtgagact tc#atctcaaa  46740aaaaaaaaaa aaaaaaaaaa aaagaacaga aatttatttc tcactgttct ag#aggctgga  46800aagtccaaga tcaaggcact gtaggctgtt gtccagtgag tatatttggt ct#ccaagtta  46860gtgccttgtc gctgcatcct ccagataggg caaatgctgt gtccttacat gg#tggaaggg  46920tagaagagca aacgggcctg actgattccc tctagctcct ttataagggc at#tcatctct  46980gtccttgtgt cctaatcaca cgctaaaggt ggctaaaggc cccacctctt aa#tactgttg  47040cattggggat aaagtttcaa catgaattat gaagagaata caaacattta aa#ccacaaca  47100agtcctttgc ccactttttt tttggagacc gagtctcact ctgttgccca gg#ctggaatg  47160cagtggcttg atcctggctc attgcaacct ccacctcctg ggttcaagca at#tctcctgc  47220ctcagcttcc caagtagctg ggattacagg tgtgcactac cacacccagc ta#attttgta  47280tatttagtag agacagggtt ttaccatgtt agccaggctg atctcgaact ct#cgacttct  47340ggtgatccac ctgcctcagc ctcccaaagt gctgagatta caggcgtgag cc#accgtgcc  47400cggccctttg cccactgttt aatggggttg tcttcttgct attgagttcc tt#atatattt  47460tttatattaa ccccttatca aatgtatggc ttgcaaatat tttctcccat cg#taggttgt  47520ctcttcactc taatgattgt ttcctttgct ctgaagacac tttttagttt ta#tttattcc  47580catttgtcta ttttcacatt tgttgcctat aagcaggtta gaaaattata ca#gattataa  47640atagttcctg aatttgtgtt ttactaaacg tagcctacac agatgaaaac ag#gaaagcta  47700cacttcagaa tctgtgatat ttgatgtcag aagtgcatcc ctgaaagcaa tg#ggtccatt  47760ctaaatctcc taacctctaa ccataatttg ttctatattt atcctgagat ct#cactctta  47820ggaataaaaa cacattgaga agtcctgagt ctctatttta ctatttttct ga#agtgcctg  47880tagtgtgtgt gtttacatct aaataatagc tgtcaccact ttctgatcaa tt#ttaaaaac  47940taattttaaa taagtgtttt tcataaataa tcctggattt agttctaaaa tc#agaataaa  48000ctatgcaaac tttgaatcca ttaatcaaaa tgcttttagt ttccattcca ac#aaaggcag  48060ataaacagcc ccttcagacc actgtggttt gaaacatagc actcactggc tg#ccttttaa  48120gagccttcag ggagggagca aaacaacaat ttttggtttt cagtttccca ga#cagtgaag  48180gagagattta gtaattttct caagtgaaaa agaattcaat aacttgcaaa ta#gaaactga  48240gatcaaattt ccaaataaag tatattgaat ttttgtttaa acttttaaaa tc#tcaagctt  48300aaagctttga acataagatt aaaaaaactt tttttagtat ccattttgtt gg#ctttagtt  48360aaatatcata caaagtaacc aaccatctgg taactttcac cttagagaaa ac#atgatagt  48420ggttgtcacc tatttcttct attgttttct cttcattatc tttgctttct tt#tcactgca  48480ctttgccagc caacagagga tgtatgggta catgtgactc acacccactt gt#ttacacat  48540gcatctgtgc aaatacataa gatggtaggt taaaaaaaga agaattagtt tc#ttgtcccc  48600tggccttctc ccacaaaaga agaattagtc cagttggttt ttcaaaatgg at#tccaggat  48660tcttagtgtt ccctcgggct cagggtggtt gataggaaaa gcctataatc ct#ctcagtca  48720cttttcagtt tgtttaggga atggatcaaa gaaggaagat tttactgggt gg#catgattt  48780ttttattata tgagggaaaa tagcacttca ctgtcttttg tttaaagaca ag#cttaacag  48840atgctaaaaa gtacatctct cagccagatt cctagtcaac aagctgatag ac#actaagat  48900tctggattct tcattgatta tattcagtca ttgttgggca attgactccc tg#ccataata  48960attgggccag tatctataac cagcatttta cagatggatt cgctagactc tt#tctgtaag  49020agatgtttct aaaaagagtt atagtgagat atgcttctaa gaaaagttat ac#tgtagtag  49080tgtaatgaaa gctactagtg ttttattagt atttcacaag aacaatgtta ct#ctgtctcc  49140catatataac tgtctatggg cttttatgat tattctttaa aaaaaaaaaa ta#ctaaggta  49200atgcctaccg gggaactcat ggtgctggct tcatccaaag tctgagctgt tt#tggcttta  49260tactccgaaa gactttattt tcatacatct taactaaaaa ctggggcttt aa#attggtca  49320ttcaaggcca ggcgcggttg ctcatgcctg aaatcccagc actttgggag gc#cgaggtgg  49380gcagatcacg aggtcaggag attgagacca tcctggccaa cacggtgaaa cc#ccgtctct  49440actaaaaata caacaacaac aacaacaaaa atagccaggc gtggtggctt gc#atctgtaa  49500tcccagctac tcaggaggct gaggcaggag aatggtgtga acctgggagg ca#gagcttgc  49560agtgagccga gatcgcatca ctgcactcca gcctgggcga cagagcgaga ct#ccgtctca  49620aaaaaaaaaa acatcggtaa ttcaaagcat agaccagccc tttttcaagt ga#tgttgttc  49680ccatgacaat ccatcagtga aaaaccaaat accatattcc aagctgctag tc#acagagaa  49740aacaagcaga tgagatgaat gtaatagaaa agactagagt tagttttggg gt#catcttta  49800gccaacattc cattgcctga agctcagtaa tctgaatcct ttttaatttg ag#cacatcag  49860ggaacagctg aatacccatg ctgaggcata atttaagctg tcaagtgtct cc#tgtcaata  49920tacatgtggt catctgatgc aaggcaaaga gacagtcact cctgcttctt ta#tatcccta  49980gctcccaaca tggtgtccta atgcatgata atcatgcagt aaatgttcag tg#atgagaac  50040atgactttga gcaaggctgt atgatctgcc tcagaacaag tgagtcagta ag#aatgcagg  50100ccccggacca taggaatgta ttacagtttt gcccaagaaa ccacaaacgt tg#gaaacact  50160caagtttctt tctcgtatac atcagctggt gtcatgcaat gggacatacc at#ctgacgct  50220tccctgttct tccctgattt gtcctgcatg tctccaatac ctctttccaa cc#acctcatc  50280tccccacctc acctttcttt ttctttgttt ggctttatat aggtgctggg ag#tccctaca  50340gaggatactt ggccgggagt ctccaagcta cctaactaca atccaggtaa ta#ttgatctg  50400agcttctgaa tactctgaga attagtaatg taaggagagc attggccacg ct#aacagggc  50460gttcttgtat tgtgaactca gcggcaaaga tgggtgtaga ggaatttcta ca#ttcatata  50520ttccctgact aatctttgta tgaggaagac actgaaagag tagctgaggt ta#gaccagtt  50580ccccagctct gtaaaacaca agtagcaagc tgaatagaat ttgaaatgac ta#ttactgtg  50640gattccacat ccattgtcaa atacccaatg gctcaaaaga acaactcaaa ag#atgggctc  50700acttttgggc cccctgactg tcataagtgt attgattagt attgaattgc at#atgtataa  50760aaagaaagct aatgcaacag aacagaggta gaggctcgct aggcctagga ca#tgccaagt  50820aagctgtctg taggttatac ttactaagag ttcattcatt gcctgtaaac ct#gacacttg  50880gtcattgtct ctcacacatt tcatctttca agactggctt ctgggatcga tt#tagaagtg  50940ctggaagtgt tatccatggg ggaattcttt gagaagctgt cgcagggcca ca#tcagaggg  51000atcagattaa gcagtagtca cttcaaggat gttgagacag aggggaggag ac#aggcactg  51060aactacagga tgaaggatca tattagaagc tgaagaagca aataaagccc at#gccaaagc  51120tgagctctca ctggcagggt tgaaggggag gtagaaaggt acagataacg ac#aagattag  51180ggtggatatg ctccaagcca gatttttcta gtctttatgg tcttacattg tt#ccattact  51240aaaaatgaaa tcttcccaaa ttgttgtcct tacttttttt tttttttttt tg#agatggag  51300ttttgctctt atcgcccagg ctggagtgca gtggcacgat ctcggctcac tg#caacctcc  51360acctcctggg ttcaagcaat tctcctgcct cagcctcccc aagtagctgg ga#ctacaggc  51420acccgccacc atgcccagct aattttttgt atttttagta gagatgaggt tt#caccatgt  51480tggccaggct ggtctcgaac tcctgacctc aggtgatcca cttgcttcag ct#tcccaaaa  51540tgctgggatt acaggcatga gccagcgcgc ctggcctgtt gtccttacta ac#tttggtat  51600gagattatcc tggaagggtt tcctgagagc aagaaattgt aggtagagtt aa#aatgtgat  51660taaagaagag aataaaatac atagggagct ggggactctt tttcttattt tc#tttagcat  51720ccaatacttt tgcttacagc tatccatagg gatctggcat cttgaaccac ca#ggattatg  51780gaagccctac agcaagctaa agactaactg taaagtcctt tcagctgctt tg#tgaatggt  51840tatatctatt gctaaaaggc cttaatatca tttgcaaata gtttatgatt tc#taactatt  51900tttctagagt ttaacacgtg agaaaaatgc tactctctgg tcacaggact ta#gaatagtg  51960cctatttcca ttggtctgag atagagaaaa aagaacaagt ttcttgtgga gc#cgtggtcc  52020agtctgcaaa ttgctcctat ctccagttgc catggtttcc aggagaacgt gg#ctctcatc  52080ttttcctgcc ctgcctgtac ttctccctgt ccactctgtt ctctattttc cc#tcagcttc  52140ctaactgagg atgccagcag aagtttagag tcacagatgg attgtaggaa ac#aatttgga  52200tgatgccaat acaaagctac tgtggtgggc atatgctgct cccccaaact tc#agacattt  52260gggtttcagg ttggtccagg caatcaacag tgatccttaa tacaaaatgt ct#tggtgaga  52320gcaataatca agaaacttgg ccaaagtgct tccctgccag attgtgtgct ta#ataagata  52380actgggttcc aataaaacag agaaaatatg ttacatttta aaaaattttc tg#ttgtttca  52440aaacaatgtg cagtttttct atataagaag aaaagtctcc aggcccaaca tc#catagggc  52500tcatcatcca ttgtttttct tttaagtttt caatttaatc caaataagtc aa#aaattttc  52560aggtacctac tatctgccag gtgctgtgcc gtgcgctggg gctacacaga tg#gagagggt  52620gcattcttgg atctctagtg tttgggtttg gattcattca cccacactct tt#caccagtt  52680ctctttgtta ctggggtgct catttgtgag ccctgcttcc atggcttgga ga#gtttgtgg  52740ctgtgggcca ggctgagctt atggagcaaa gggagttgga accttagcca ta#gacatgat  52800gtctaaacct ggatttggaa atcttaaaag tccagcctat cttgggccat gg#ggtcagta  52860ttattgataa ctcaatccca aggactgtgt tttaaaaggg tctccaacat ct#gcatttca  52920ggaacatcct cttacgtgag tcaataagtt ccttttgagc caccccctac cc#atccccat  52980ccctgagctg ctgtggcttc taaacacttg aatgtcagtg attaagggga gc#agaagaca  53040agctgggagc caggaaagtg tcacagatga gcaccgtgtc agcagcattc tg#gatgagct  53100tcccattcct ttccttttca ttctaagtag tcctaggagc ccccaaactt tg#aatcagcc  53160agtacaattt tgagggagtc cagttgtccg gaacttggga gaaccatcca gt#gtccatct  53220acacccatgc ctccatttct aggccttatc tggacacctc taggaggaca gc#aaagtttc  53280catttgtaca gcttttaaaa agtcacctga tgctgaccca gtcggatttc tc#          53332 <210> SEQ ID NO 4 <211> LENGTH: 1308 <212> TYPE: DNA<213> ORGANISM: Human <400> SEQUENCE: 4atgggtcaag agctgtgtgc aaagactgta cagcctggat gcagctgcta cc#attgttca     60gagggaggcg aggcacacag ctgtcggagg agtcagcctg agaccacgga gg#ctgcgttc    120aagctaacag acctaaaaga agcatcatgt tccatgactt catttcaccc ca#ggggactt    180caagctgccc gtgcccagaa gttcaagagt aaaaggccac ggagtaacag tg#attgtttt    240caggaagagg atctgaggca gggttttcag tggaggaaga gcctcccttt tg#gggcagcc    300tcatcttact tgaacttgga gaagctgggt gaaggctctt atgcgacagt tt#acaagggg    360attagcagaa taaatggaca actagtggct ttaaaagtca tcagcatgaa tg#cagaggaa    420ggagtcccat ttacagctat ccgagaagct tctctcctga agggtttgaa ac#atgccaat    480attgtgctcc tgcatgacat aatccacacc aaagagacac tgacattcgt tt#ttgaatac    540atgcacacag acctggccca gtatatgtct cagcatccag gagggcttca tc#ctcataat    600gtcagacttt tcatgtttca acttttgcgg ggcctggcgt acatccacca cc#aacacgtt    660cttcacaggg acctgaaacc tcagaactta ctcatcagtc acctgggaga gc#tcaaactg    720gctgattttg gtcttgcccg ggccaagtcc attcccagcc agacatactc tt#cagaagtc    780gtgaccctct ggtaccggcc ccctgatgct ttgctgggag ccactgaata tt#cctctgag    840ctggacatat ggggtgcagg ctgcatcttt attgaaatgt tccagggtca ac#ctttgttt    900cctggggttt ccaacatcct tgaacagctg gagaaaatct gggaggtgct gg#gagtccct    960acagaggata cttggccggg agtctccaag ctacctaact acaatccaga at#ggttccca   1020ctgcctacgc ctcgaagcct tcatgttgtc tggaacaggc tgggcagggt tc#ctgaagct   1080gaagacctgg cctcccagat gctaaaaggc tttcccagag accgcgtctc cg#cccaggaa   1140gcacttgttc atgattattt cagcgccctg ccatctcagc tgtaccagct tc#ctgatgag   1200gagtctttgt ttacagtttc aggagtgagg ctaaagccag aaatgtgtga cc#ttttggcc   1260 tcctaccaga aaggtcacca cccagcccag tttagcaaat gctggtga  #              1308 <210> SEQ ID NO 5 <211> LENGTH: 435 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 5Met Gly Gln Glu Leu Cys Ala Lys Thr Val Gl #n Pro Gly Cys Ser Cys 1               5   #                10   #                15Tyr His Cys Ser Glu Gly Gly Glu Ala His Se #r Cys Arg Arg Ser Gln            20       #            25       #            30Pro Glu Thr Thr Glu Ala Ala Phe Lys Leu Th #r Asp Leu Lys Glu Ala        35           #        40           #        45Ser Cys Ser Met Thr Ser Phe His Pro Arg Gl #y Leu Gln Ala Ala Arg    50               #    55               #    60Ala Gln Lys Phe Lys Ser Lys Arg Pro Arg Se #r Asn Ser Asp Cys Phe65                   #70                   #75                   #80Gln Glu Glu Asp Leu Arg Gln Gly Phe Gln Tr #p Arg Lys Ser Leu Pro                85   #                90   #                95Phe Gly Ala Ala Ser Ser Tyr Leu Asn Leu Gl #u Lys Leu Gly Glu Gly            100       #           105       #           110Ser Tyr Ala Thr Val Tyr Lys Gly Ile Ser Ar #g Ile Asn Gly Gln Leu        115           #       120           #       125Val Ala Leu Lys Val Ile Ser Met Asn Ala Gl #u Glu Gly Val Pro Phe    130               #   135               #   140Thr Ala Ile Arg Glu Ala Ser Leu Leu Lys Gl #y Leu Lys His Ala Asn145                 1 #50                 1 #55                 1 #60Ile Val Leu Leu His Asp Ile Ile His Thr Ly #s Glu Thr Leu Thr Phe                165   #               170   #               175Val Phe Glu Tyr Met His Thr Asp Leu Ala Gl #n Tyr Met Ser Gln His            180       #           185       #           190Pro Gly Gly Leu His Pro His Asn Val Arg Le #u Phe Met Phe Gln Leu        195           #       200           #       205Leu Arg Gly Leu Ala Tyr Ile His His Gln Hi #s Val Leu His Arg Asp    210               #   215               #   220Leu Lys Pro Gln Asn Leu Leu Ile Ser His Le #u Gly Glu Leu Lys Leu225                 2 #30                 2 #35                 2 #40Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Il #e Pro Ser Gln Thr Tyr                245   #               250   #               255Ser Ser Glu Val Val Thr Leu Trp Tyr Arg Pr #o Pro Asp Ala Leu Leu            260       #           265       #           270Gly Ala Thr Glu Tyr Ser Ser Glu Leu Asp Il #e Trp Gly Ala Gly Cys        275           #       280           #       285Ile Phe Ile Glu Met Phe Gln Gly Gln Pro Le #u Phe Pro Gly Val Ser    290               #   295               #   300Asn Ile Leu Glu Gln Leu Glu Lys Ile Trp Gl #u Val Leu Gly Val Pro305                 3 #10                 3 #15                 3 #20Thr Glu Asp Thr Trp Pro Gly Val Ser Lys Le #u Pro Asn Tyr Asn Pro                325   #               330   #               335Glu Trp Phe Pro Leu Pro Thr Pro Arg Ser Le #u His Val Val Trp Asn            340       #           345       #           350Arg Leu Gly Arg Val Pro Glu Ala Glu Asp Le #u Ala Ser Gln Met Leu        355           #       360           #       365Lys Gly Phe Pro Arg Asp Arg Val Ser Ala Gl #n Glu Ala Leu Val His    370               #   375               #   380Asp Tyr Phe Ser Ala Leu Pro Ser Gln Leu Ty #r Gln Leu Pro Asp Glu385                 3 #90                 3 #95                 4 #00Glu Ser Leu Phe Thr Val Ser Gly Val Arg Le #u Lys Pro Glu Met Cys                405   #               410   #               415Asp Leu Leu Ala Ser Tyr Gln Lys Gly His Hi #s Pro Ala Gln Phe Ser            420       #           425       #           430 Lys Cys Trp        435 <210> SEQ ID NO 6 <211> LENGTH: 240 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 6Phe Gly Lys Ala Asp Ser Tyr Glu Lys Leu Gl #u Lys Leu Gly Glu Gly 1               5   #                10   #                15Ser Tyr Ala Thr Val Tyr Lys Gly Lys Ser Ly #s Val Asn Gly Lys Leu            20       #            25       #            30Val Ala Leu Lys Val Ile Arg Leu Gln Glu Gl #u Glu Gly Thr Pro Phe        35           #        40           #        45Thr Ala Ile Arg Glu Ala Ser Leu Leu Lys Gl #y Leu Lys His Ala Asn    50               #    55               #    60Ile Val Leu Leu His Asp Ile Ile His Thr Ly #s Glu Thr Leu Thr Leu65                   #70                   #75                   #80Val Phe Glu Tyr Val His Thr Asp Leu Cys Gl #n Tyr Met Glu Gln His                85   #                90   #                95Pro Gly Gly Leu His Pro Asp Asn Val Lys Le #u Phe Leu Phe Gln Leu            100       #           105       #           110Leu Arg Gly Leu Ser Tyr Ile His Gln Arg Ty #r Ile Leu His Arg Asp        115           #       120           #       125Leu Lys Pro Gln Asn Leu Leu Ile Ser Asp Th #r Gly Glu Leu Lys Leu    130               #   135               #   140Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Va #l Pro Ser His Thr Tyr145                 1 #50                 1 #55                 1 #60Ser Asn Glu Val Val Thr Leu Trp Tyr Arg Pr #o Pro Asp Val Leu Leu                165   #               170   #               175Gly Ser Thr Glu Tyr Ser Thr Cys Leu Asp Me #t Trp Gly Val Gly Cys            180       #           185       #           190Ile Phe Val Glu Met Ile Gln Gly Val Ala Al #a Phe Pro Gly Met Lys        195           #       200           #       205Asp Ile Gln Asp Gln Leu Glu Arg Ile Phe Le #u Val Leu Gly Thr Pro    210               #   215               #   220Asn Glu Asp Thr Trp Pro Gly Val His Ser Le #u Pro His Phe Lys Pro225                 2 #30                 2 #35                 2 #40<210> SEQ ID NO 7 <211> LENGTH: 245 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 7Phe Gly Lys Ala Asp Ser Tyr Glu Lys Leu Gl #u Lys Leu Gly Glu Gly 1               5   #                10   #                15Ser Tyr Ala Thr Val Tyr Lys Gly Lys Ser Ly #s Val Asn Gly Lys Leu            20       #            25       #            30Val Ala Leu Lys Val Ile Arg Leu Gln Glu Gl #u Glu Gly Thr Pro Phe        35           #        40           #        45Thr Ala Ile Arg Glu Ala Ser Leu Leu Lys Gl #y Leu Lys His Ala Asn    50               #    55               #    60Ile Val Leu Leu His Asp Ile Ile His Thr Ly #s Glu Thr Leu Thr Leu65                   #70                   #75                   #80Val Phe Glu Tyr Val His Thr Asp Leu Cys Gl #n Tyr Met Asp Lys His                85   #                90   #                95Pro Gly Gly Leu His Pro Asp Asn Val Lys Le #u Phe Leu Phe Gln Leu            100       #           105       #           110Leu Arg Gly Leu Ser Tyr Ile His Gln Arg Ty #r Ile Leu His Arg Asp        115           #       120           #       125Leu Lys Pro Gln Asn Leu Leu Ile Ser Asp Th #r Gly Glu Leu Lys Leu    130               #   135               #   140Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Va #l Pro Ser His Thr Tyr145                 1 #50                 1 #55                 1 #60Ser Asn Glu Val Val Thr Leu Trp Tyr Arg Pr #o Pro Asp Val Leu Leu                165   #               170   #               175Gly Ser Thr Glu Tyr Ser Thr Cys Leu Asp Me #t Trp Gly Val Gly Cys            180       #           185       #           190Ile Phe Val Glu Met Ile Gln Gly Val Ala Al #a Phe Pro Gly Met Lys        195           #       200           #       205Asp Ile Gln Asp Gln Leu Glu Arg Ile Phe Le #u Val Leu Gly Thr Pro    210               #   215               #   220Asn Glu Asp Thr Trp Pro Gly Val His Ser Le #u Pro His Phe Lys Pro225                 2 #30                 2 #35                 2 #40Glu Arg Phe Thr Leu                 245 <210> SEQ ID NO 8<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 8 Phe Gly Lys Ala Asp Ser Tyr Glu Lys Leu Gl#u Lys Leu Gly Glu Gly  1               5   #                10  #                15 Ser Tyr Ala Thr Val Tyr Lys Gly Lys Ser Ly#s Val Asn Gly Lys Leu             20       #            25      #            30 Val Ala Leu Lys Val Ile Arg Leu Gln Glu Gl#u Glu Gly Thr Pro Phe         35           #        40          #        45 Thr Ala Ile Arg Glu Ala Ser Leu Leu Lys Gl#y Leu Lys His Ala Asn     50               #    55              #    60 Ile Val Leu Leu His Asp Ile Ile His Thr Ly#s Glu Thr Leu Thr Leu 65                   #70                  #75                   #80 Val Phe Glu Tyr Val His Thr Asp Leu Cys Gl#n Tyr Met Glu Gln His                 85   #                90  #                95 Pro Gly Gly Leu His Pro Asp Asn Val Lys Le#u Phe Leu Phe Gln Leu             100       #           105      #           110 Leu Arg Gly Leu Ser Tyr Ile His Gln Arg Ty#r Ile Leu His Arg Asp         115           #       120          #       125 Leu Lys Pro Gln Asn Leu Leu Ile Ser Asp Th#r Gly Glu Leu Lys Leu     130               #   135              #   140 Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Va#l Pro Ser His Thr Tyr 145                 1 #50                 1#55                 1 #60 Ser Asn Glu Val Val Thr Leu Trp Tyr Arg Pr#o Pro Asp Val Leu Leu                 165   #               170  #               175 Gly Ser Thr Glu Tyr Ser Thr Cys Leu Asp Me#t Trp Gly Val Gly Cys             180       #           185      #           190 Ile Phe Val Glu Met Ile Gln Gly Val Ala Al#a Phe Pro Gly Met Lys         195           #       200          #       205 Asp Ile Gln Asp Gln Leu Glu Arg Ile Phe Le#u Val Leu Gly Thr Pro     210               #   215              #   220 Asn Glu Asp Thr Trp Pro Gly Val His Ser Le#u Pro His Phe Lys Pro 225                 2 #30                 2#35                 2 #40 Glu Arg Phe Thr Val Tyr Ser Ser Lys Ser Le#u Arg Gln Ala Trp Asn                 245   #               250  #               255 Lys Leu Ser Tyr Val Asn His Ala Glu Asp Le#u Ala Ser Lys Leu Leu             260       #           265      #           270 Gln Cys Ser Pro Lys Asn Arg Leu Ser Ala Gl#n Ala Ala Leu Ser His         275           #       280          #       285 Glu Tyr Phe Ser Asp Leu Pro Pro Arg Leu Tr#p Glu Leu Thr Asp Met     290               #   295              #   300 Ser Ser Ile Phe Thr Val Pro Asn Val Arg Le#u Gln Pro Glu Ala Gly 305                 3 #10                 3#15                 3 #20 Glu Ser Met Arg Ala Phe Gly Lys Asn Asn                325   #               330 <210> SEQ ID NO 9<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 9 Phe Gly Lys Ala Asp Ser Tyr Glu Lys Leu Gl#u Lys Leu Gly Glu Gly  1               5   #                10  #                15 Ser Tyr Ala Thr Val Tyr Lys Gly Lys Ser Ly#s Val Asn Gly Lys Leu             20       #            25      #            30 Val Ala Leu Lys Val Ile Arg Leu Gln Glu Gl#u Glu Gly Thr Pro Phe         35           #        40          #        45 Thr Ala Ile Arg Glu Ala Ser Leu Leu Lys Gl#y Leu Lys His Ala Asn     50               #    55              #    60 Ile Val Leu Leu His Asp Ile Ile His Thr Ly#s Glu Thr Leu Thr Leu 65                   #70                  #75                   #80 Val Phe Glu Tyr Val His Thr Asp Leu Cys Gl#n Tyr Met Asp Lys His                 85   #                90  #                95 Pro Gly Gly Leu His Pro Asp Asn Val Lys Le#u Phe Leu Phe Gln Leu             100       #           105      #           110 Leu Arg Gly Leu Ser Tyr Ile His Gln Arg Ty#r Ile Leu His Arg Asp         115           #       120          #       125 Leu Lys Pro Gln Asn Leu Leu Ile Ser Asp Th#r Gly Glu Leu Lys Leu     130               #   135              #   140 Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Va#l Pro Ser His Thr Tyr 145                 1 #50                 1#55                 1 #60 Ser Asn Glu Val Val Thr Leu Trp Tyr Arg Pr#o Pro Asp Val Leu Leu                 165   #               170  #               175 Gly Ser Thr Glu Tyr Ser Thr Cys Leu Asp Me#t Trp Gly Val Gly Cys             180       #           185      #           190 Ile Phe Val Glu Met Ile Gln Gly Val Ala Al#a Phe Pro Gly Met Lys         195           #       200          #       205 Asp Ile Gln Asp Gln Leu Glu Arg Ile Phe Le#u Val Leu Gly Thr Pro     210               #   215              #   220 Asn Glu Asp Thr Trp Pro Gly Val His Ser Le#u Pro His Phe Lys Pro 225                 2 #30                 2#35                 2 #40 Glu Arg Phe Thr Leu Tyr Ser Ser Lys Asn Le#u Arg Gln Ala Trp Asn                 245   #               250  #               255 Lys Leu Ser Tyr Val Asn His Ala Glu Asp Le#u Ala Ser Lys Leu Leu             260       #           265      #           270 Gln Cys Ser Pro Lys Asn Arg Leu Ser Ala Gl#n Ala Ala Leu Ser His         275           #       280          #       285 Glu Tyr Phe Ser Asp Leu Pro Pro Arg Leu Tr#p Glu Leu Thr Asp Met     290               #   295              #   300 Ser Ser Ile Phe Thr Val Pro Asn Val Arg Le#u Gln Pro Glu Ala Gly 305                 3 #10                 3#15                 3 #20 Glu Ser Met Arg Ala Phe Gly Lys Asn Asn                325   #               330 <210> SEQ ID NO 10<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 10 Phe Gly Lys Ala Asp Ser Tyr Glu Lys Leu Gl#u Lys Leu Gly Glu Gly  1               5   #                10  #                15 Ser Tyr Ala Thr Val Tyr Lys Gly Lys Ser Ly#s Val Asn Gly Lys Leu             20       #            25      #            30 Val Ala Leu Lys Val Ile Arg Leu Gln Glu Gl#u Glu Gly Thr Pro Phe         35           #        40          #        45 Thr Ala Ile Arg Glu Ala Ser Leu Leu Lys Gl#y Leu Lys His Ala Asn     50               #    55              #    60 Ile Val Leu Leu His Asp Ile Ile His Thr Ly#s Glu Thr Leu Thr Leu 65                   #70                  #75                   #80 Val Phe Glu Tyr Val His Thr Asp Leu Cys Gl#n Tyr Met Asp Lys His                 85   #                90  #                95 Pro Gly Gly Leu His Pro Asp Asn Val Lys Le#u Phe Leu Phe Gln Leu             100       #           105      #           110 Leu Arg Gly Leu Ser Tyr Ile His Gln Arg Ty#r Ile Leu His Arg Asp         115           #       120          #       125 Leu Lys Pro Gln Asn Leu Leu Ile Ser Asp Th#r Gly Glu Leu Lys Leu     130               #   135              #   140 Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Va#l Pro Ser His Thr Tyr 145                 1 #50                 1#55                 1 #60 Ser Asn Glu Val Val Thr Leu Trp Tyr Arg Pr#o Pro Asp Val Leu Leu                 165   #               170  #               175 Gly Ser Thr Glu Tyr Ser Thr Cys Leu Asp Me#t Trp Gly Val Gly Cys             180       #           185      #           190 Ile Phe Val Glu Met Ile Gln Gly Val Ala Al#a Phe Pro Gly Met Lys         195           #       200          #       205 Asp Ile Gln Asp Gln Leu Glu Arg Ile Phe Le#u Val Leu Gly Thr Pro     210               #   215              #   220 Asn Glu Asp Thr Trp Pro Gly Val His Ser Le#u Pro His Phe Lys Pro 225                 2 #30                 2#35                 2 #40 Glu Arg Phe Thr Val Tyr Asn Ser Lys Ser Le#u Arg Gln Ala Trp Asn                 245   #               250  #               255 Lys Leu Ser Tyr Val Asn His Ala Glu Asp Le#u Ala Ser Lys Leu Leu             260       #           265      #           270 Gln Cys Ser Pro Lys Asn Arg Leu Ser Ala Gl#n Ala Ala Leu Ser His         275           #       280          #       285 Glu Tyr Phe Ser Asp Leu Pro Pro Arg Leu Tr#p Glu Leu Thr Asp Met     290               #   295              #   300 Ser Ser Ile Phe Thr Val Pro Asn Val Arg Le#u Gln Pro Glu Ala Gly 305                 3 #10                 3#15                 3 #20 Glu Ser Met Arg Ala Phe Gly Lys Asn Asn                325   #               330

That which is claimed is:
 1. An isolated polypeptide having an aminoacid sequence consisting of SEQ ID NO:2.
 2. An isolated polypeptidehaving an amino acid sequence comprising SEQ ID NO:2.
 3. A compositioncomprising the polypeptide of claim 1 and a carrier.
 4. A compositioncomprising the polypeptide of claim 2 and a carrier.